Compositions and methods for regulating sas1r

ABSTRACT

The present invention provides compositions and methods useful for regulating fertilization and for use as a contraceptive based on the discovery herein of an oocyte specific protein, SAS1R (Sperm Acrosomal SLLP1 Receptor), which is a sperm protein receptor. Six SAS1R variants, including the full length SAS1R, were identified. mSLLP1 and SAS1R co-localized to oocytes and to acrosomes of acrosome-reacted sperm. Interactions between mSLLP1 and SAS1R were demonstrated by far-western analysis, in a yeast two-hybrid system under stringent selection conditions, and by immunoprecipitation of SAS1R by anti-mSLLP1 as well as the converse. Purified recombinant SAS1R was found to have protease activity, to inhibit fertilization in-vitro, and to induce an immune response in females. Together, the results suggest SAS1R is a proteolytically active, oocyte and early embryo specific oolemmal metalloprotease receptor for the sperm intra-acrosomal ligand SLLP1 and is a target for regulating fertilization and as a contraceptive.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/670,640, filed Aug. 7, 2017, now abandoned, which is a divisional ofU.S. patent application Ser. No. 13/479,167, filed May 23, 2012, nowU.S. Pat. No. 9,803,012, which is a divisional of U.S. patentapplication Ser. No. 12/613,947, filed Nov. 6, 2009, now abandoned,which is a continuation-in-part of U.S. patent application Ser. No.11/915,225, filed Nov. 24, 2008, now abandoned, which claims the benefitof priority to International Application No. PCT/US2006/005970, filed onFeb. 21, 2006, which is entitled to priority pursuant to 35 U.S.C. §119(e) to U.S. Provisional Patent Application Ser. Nos. 60/655,562,filed Feb. 23, 2005, and 60/689,181, filed Jun. 10, 2005, and thisapplication is further entitled priority to U.S. Provisional PatentApplication Ser. Nos. 61/111,903, filed Nov. 6, 2008, 61/225,790, filedJul. 15, 2009, 61/243,411 filed Sep. 17, 2009, and 61/244,543, filedSep. 22, 2009, the entire disclosures of all of which are hereinincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. R03HD055129 awarded by the NIH and D43 TW/HD 00654 from the FogartyInternational Center. The government has certain rights in theinvention.

SEQUENCE LISTING

Incorporated by reference herein in its entirety is a computer-readablesequence listing submitted via EFS-Web and identified as follows: One(53,219 byte ASCII (Text)) file named “20220220 Sequence_Listing.txt”created on Feb. 20, 2022.

BACKGROUND

Among the events of fertilization, few are more important, yetenigmatic, than interactions between the sperm and egg membranes.Although several sperm proteins that bind to mammalian oocytes have beenidentified, there has been less success in identifying oolemmalreceptors for sperm ligands. The unique testis-specific c lysozyme-like,intra-acrosomal protein SLLP1, was reported to lack bacteriolyticactivity (1); localize to mouse sperm acrosomal membranes, and haveoolemma binding properties (2). SLLP1 antibody and recombinant (r) SLLP1were noted to block fertilization and sperm-egg binding in mice,suggesting that the protein may play a role in sperm/egg adhesion.

Molecules posited to be involved in sperm-oolemmal binding and fusioninclude the ADAM family ligands and their oocyte integrin receptors(3-6). However, gene targeting studies have demonstrated that the spermADAMs including fertilin α (ADAM1), fertilino (ADAM2) and cyritestin(ADAM3) are important primarily for the process of zona pellucidabinding rather than for gamete fusion (7-9). Attention has also focusedon tetraspanins (e.g., CD9, CD81), on GPI-anchored proteins, and onPIG-A which are expressed on oocytes. Data suggest that these proteinsare important for the sperm-oocyte fusion step, but not for the bindingprocess (10-12). Although CD9^(−/−) female mice produced eggs thatmatured normally, sperm-egg fusion failed in these animals (13, 14).Targeted disruption of CD81 resulted in a 40% reduction in fertility ofonly female mice while mice lacking both CD9 and CD81 were completelyinfertile, indicating their complementary roles in sperm-egg fusion(15). It is noteworthy that sperm ligands that interact with oolemmaltetraspanins have not been identified. Several sperm membrane ligandshave been implicated in fusion with the oocyte although their oolemmalreceptors are unknown. Epididymal protein DE (CRISP1) has beenimplicated in sperm-oocyte fusion (16) and a specific binding regionwithin CRISP1 was mapped, however CRISP1 knockout male and female miceshowed no differences in fertility compared to controls (17). Recently,Izumo, an Ig-domain molecule localized within the acrosome was shown tobe essential for sperm-egg fusion (18) although its oolemmal receptor isstill unknown.

Moreover, there has been a concerted effort to identify biomarkers anddifferentiation antigens that are specific to the sperm or the egg inorder to target these cells for contraceptive purposes, including drugand vaccine development. These approaches have included monoclonalantibodies directed a the gametes, proteomic, transcriptomic and genomicapproaches (Nass et al., Nat Rev Drug Discov. 2004 October;3(10):885-90); Nass et al., Science. 2004 Mar. 19; 303(5665):1769-71;Nass et al., National Academy Press, p 27-77, (2004), Contraception.2008 October; 78 (4 Suppl):S28-35. Epub 2008 Aug. 22; Aitken et al.,Contraception. 2008 October; 78 (4 Suppl):S18-22. Epub 2008 Jun. 12.)

Candidates proteins for contraceptive targeting must meet selectivecriteria including 1) restriction of the protein to the gamete; 2) anessential role for the protein in key stages of gametogenesis,fertilization or implantation; 3) accessibility of the drug target atthe cell surface or within a select window of differentiation; 4)structural domains amenable to drug targeting (drugability); and 5) therestriction of the target to selected stages of gamete differentiationthat permit targeted drug action and contraceptive reversibility in thecase of human applications. Until now, few proteins have been elucidatedthat meet all of these criteria.

There is a long felt need in the art to identify both sperm and oolemmaspecific interacting proteins involved in the process of fertilizationand to find methods to regulate these interactions to regulate fertilityand contraception. The present invention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful ascontraceptive vaccines, as methods of inducing an immune response, as acontraceptive drug target for selectively affecting maturing oocyteswhile sparing young oogonia that are naked, or within primordial orprimary follicles; and as a method of modulating fertilization andfertility. The compositions comprise at least one mammalian egg protein,or a homolog, derivative, or fragment thereof, or at least one isolatednucleic acid comprising a nucleic acid sequence encoding an egg protein.The compositions are particularly useful as contraceptives for,including, but not limited to, humans, dogs, cats, and mice.

The compositions and methods of the invention are based on theunexpected results disclosed herein that SAS1R (Sperm Acrosomal SLLP1Receptor) is, inter alia, an immunogenic oocyte stage specificmetalloprotease that binds with the sperm protein SLLP1, furthermorethat inhibition of the protein or its interaction with SLLP1 inhibitsfertilization, and because of its specific stage specific expression infollicles its use as a contraceptive target will spare oocyte stemcells. Furthermore, these biological and biochemical properties of SAS1Rallow for its use as a contraceptive target and its use as acontraceptive vaccine, both in a reversible manner, as well as methodsof identifying contraceptive agents.

A contraceptive drug target, such as the one disclosed herein, fulfillsessential criteria for drug development including functioning infertilization and being specific to oocytes in secondary and subsequentfollicular stages such that drug targeting of this protein will notaffect stem germ cells including naked, primordial and primary oocytes.

In one aspect, the composition comprises a cocktail or mixture of two ormore different egg proteins or two or more different isolated nucleicacids comprising nucleic acid sequences encoding different egg proteins,which can be used as a contraceptive vaccine or to induce an immuneresponse directed against the egg proteins.

With knowledge of oolemmal receptors limited and with an eye to thedesign of molecular strategies for contraception, it is necessary toidentify both sperm and oolemmal specific interacting proteins involvedin the process of fertilization. The present application presentsseveral lines of evidence characterizing the molecular, biochemical, andfunctional properties of the SLLP1 receptor, SAS1R (Sperm AcrosomalSLLP1 Receptor), their interactions, and demonstrating the timing andpattern of SAS1R expression in oocytes and early embryos. SAS1R appearsto be the only oocyte and early embryo specific membrane receptor for asperm ligand that has been identified to date in mammalianfertilization. Although sperm membrane metalloproteases have beenpreviously characterized, SAS1R is the first oolemmal metalloproteaseimplicated in sperm-oolemma binding prior to sperm-egg fusion and it isdisclosed herein as being capable of inducing an immune response infemales. These properties support consideration of SAS1R as a candidatecontraceptive drug or vaccine target.

The present invention provides compositions and methods useful forinhibiting the interaction of SLLP1 with SAS1R, thereby inhibitingfertilization. The present invention provides compositions and methodsuseful for inhibiting fertilization by inhibiting the interaction ofSLLP1 and SAS1R. The interaction can be inhibited various ways,including, but not limited to, the use of purified SLLP1 and SAS1Rproteins, or analogs, derivatives, homologs, or fragments thereof, toprevent binding of the sperm protein to the egg protein. The presentapplication further provides for the use of antibodies directed againstSAS1R to inhibit fertilization. In one aspect, the type of antibodyincludes, but is not limited to, a polyclonal antibody, a monoclonalantibody, a chimeric antibody, and a synthetic antibody. In one aspect,the antibody is a monoclonal antibody.

The present invention provides compositions and methods useful foridentifying regulators of SAS1R and its function. In one aspect, theregulators are inhibitors of SAS1R. Inhibitors of SAS1R includes thosewhich inhibit its interaction or binding with a sperm protein such asSLLP1, its activity as a protease, its role in fertilization, or inhibitits regulation of downstream activities included in SAS1R signaltransduction pathways. In one aspect, the inhibitor is SAS1R, or afragment or homolog of SAS1R which binds with SLLP1. In one aspect, theSAS1R fragment is an N-terminus portion of the protein. In one aspect,the N-terminus comprises about the amino terminal 121 amino acidresidues of mature SAS1R. In another aspect, the SAS1R fragment whichbinds with SLLP1 and inhibits SLLP1 interaction with an egg is aC-terminus portion of SAS1R. In one aspect, the C-terminus of the SAS1Rcomprises about the carboxy terminal 210 amino acid residues of SAS1R.One of ordinary skill in the art will appreciate that any kind ofcompound that inhibits SAS1R levels, function, or activity as describedherein, or those that are yet unknown, are encompassed by the presentinvention.

In one embodiment, the present invention provides compositions andmethods useful for determining that SAS1R functions as an activemetalloprotease, as well as for measuring that function. These methodsare useful for determining whether a test compound or molecule caninhibit SAS1R.

In one aspect, SAS1R is inhibited in an N-terminus portion of theprotein. In one aspect, the N-terminus comprises about the aminoterminal 121 amino acid residues of mature SAS1R. In another aspect, theSAS1R is inhibited in a C-terminus portion of the protein. In oneaspect, the C-terminus of the protein comprises about the carboxyterminal 210 amino acid residues of SAS1R. In one aspect, the inhibitoris an antibody directed against SAS1R. In another aspect, the inhibitoris a drug or other compound. In one aspect, the inhibitor inhibits theprotease activity of SAS1R. In another aspect, the inhibitor inhibitsthe interaction of SAS1R with SLLP1 any other sperm protein. In oneaspect, the interaction is binding.

The present inventors have surprisingly found that suitable antigens forimmunotherapeutic strategies include the egg protein SAS1R. The presentapplication discloses immunogenic compositions comprising an immunogenderived from eggs. That antigen is the SAS1R protein, as well asantigenic fragments and homologs thereof. The present invention providescompositions and methods useful for inhibiting fertilization and forcontraception. In one aspect, SAS1R, or fragments or homologs thereofwhich maintain the immunogenic activity of full length SAS1R, can beadministered to a subject to elicit an immune response against SAS1R. Inone aspect, the administration of SAS1R and fragments and homologsthereof is useful as a vaccine.

In one embodiment, the compositions and methods of the invention areuseful in mammals. In one aspect, the mammal is a human.

Together, the data disclosed herein demonstrated that SAS1R is aproteolytically active, oocyte and early embryo specific oolemmalmetalloprotease receptor for the sperm intra-acrosomal ligand SLLP1 andis a target for regulating fertilization and as a contraceptive.Surprisingly, and importantly, the enzyme is demonstrated herein to showstage specific expression in secondary and subsequent follicularoocytes, including preantral and antral follicles, rendering it asuitable target for a reversible contraceptive that will spare naked,primordial, and primary oocytes, and preserve the ovarian reserve ofstem germ cells. Therefore, the invention further encompasses thecompositions and methods for identifying compounds that inhibit SAS1R.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Expression, purification, Western and protease activityof rSAS1R. FIG. 1A: The mature protein was expressed in E. coli (L 1),purified (L 2) and stained with Coomassie Blue. All purified bands wereconfirmed as rSAS1R by anti-His Western analysis (L 3). Antibody torSAS1R recognized the recombinant (L 4) and the native SAS1R in zonaintact oocyte extracts (L 5). Native SAS1R showed microheterogeneity.FIG. 1B: Assay of protease activity of rSAS1R (♦) using a fluorescenttagged synthetic peptide as substrate. Varying concentrations ofpurified proteins were used in 100 μL assay system. rSLLP1 (▪) was usedas a negative control.

FIGS. 2A and 2B: IF localization of SAS1R in ovary and oocytes. FIG. 2A:SAS1R localized specifically to oocyte cytoplasm within primary,secondary and Graafian follicles. FIG. 2B: In unpermeabilizedzona-intact ovulated M2 oocytes, SAS1R concentrated in a dome shapedmicrovillar domain on the surface of oocyte plasma membrane. Eccentricnuclei (blue) were antipodal to the SAS1R positive domain. Panels: left,phase; middle, fluorescence; right, merge.

FIG. 3 : Confocal localization of SAS1R, before and after fertilization.Oocytes and cultured early embryos stained with SAS1R antibody (red) andwith Sytox (for nucleus, green; upper panel; merged on phase images,lower panel). SAS1R was localized uniformly in the cytoplasm of the GVoocyte where it concentrated at the cell periphery. After polar bodyformation, in M2 oocyte SAS1R localized mainly in the microvillar domainof oolemma. In fertilized oocytes (PN-II), SAS1R was located only inpunctate regions at the cell periphery. These small patches persistedfrom 2-cell to morula stages when SAS1R appeared within the PVS. SAS1Rwas virtually undetectable in blastocyst stages.

FIGS. 4A to 4C: SAS1R expression on the cell surface of transfectedCHO-K1 cells. FIG. 4A: In fixed and permeabilized cells SAS1R localizedto cytoplasmic and surface domains. FIG. 4B: Polarized localization(arrows) of SAS1R at the cell surface in fixed, unpermeabilized cellsconfirming that SAS1R is a membrane protein. FIG. 4C: Fixed andpermeabilized cells probed with SAS1R C-terminal V5 tag antibody showingits expression in the cytoplasm. Panels: left—phase, right—fluorescence.

FIG. 5 : SAS1R—SLLP1 interaction analyses by Far-Western (FW) and Y2H.(FW) Profile of purified rSAS1R stained with Coomassie used for FWanalysis (L 1). Western of rSAS1R probed with anti-his tag antibody (L2). rSAS1R blot was either overlaid (L 3) or not overlaid (L 4) withpurified soluble rSLLP1 (5 μg/ml) and probed with anti-SLLP1 monoclonalantibody. Full length SAS1R (˜51, ˜50 kD) binds to rSLLP1 however, theequal intensity C-terminal ˜25 kD protein bound rSLLP1 very weakly.(Y2H) Y2H assay showing affinity between SLLP1 and SAS1R fragments.Growth of yeast cells on high stringency plate forming blue coloniesindicated interaction between the partner proteins. (A) Positiveinteraction between SV40 large T-antigen and its murine partner p53. (B)Negative interaction between large T-antigen and human Lamin C. (C) Veryweak interaction between full length SAS1R and SLLP1 with few smallcolonies but none in 3 days (see FIG. 14 ). (D) Positive interactionbetween N-terminal SAS1R and SLLP1. (E) Positive interaction betweenC-terminal SAS1R and SLLP1.

FIGS. 6A and 6B: Co-Immunoprecipitation of SAS1R and SLLP1. Proteinswere synthesized by in vitro translation in presence of S³⁵-methionineand analyzed by SDS-PAGE (FIG. 15 ). Translated SAS1R full length (F),N-terminus (N), C-terminus (C) and p53 (53) had Myc-tag (M) whileT-antigen (T) and SLLP1 (S) had HA-tag (H). FIG. 6A:Co-Immunoprecipitation of T-antigen or SLLP1 using Myc-tag antibody frompartner proteins. FIG. 6B: Co-Immunoprecipitation of SAS1R full length,N-terminal and C-terminal proteins using HA-tag antibody from partnerprotein. The co-immunoprecipitation products were marked by arrows. TheIPs of SLLP1 and SAS1R constructs were done using HA- and Myc-tagantibodies respectively.

FIGS. 7A and 7B: Co-localization of rSLLP1 and rSAS1R in gametes. FIG.7A: M2 oocytes incubated with rSLLP1, washed and probed with SLLP1 andSAS1R antibodies. SLLP1 predominantly co-localized to the microvillarregion of the mouse oolemma marked by SAS1R localization. FIG. 7B:Capacitated sperm incubated with rSAS1R, washed and probed with bothantibodies. SAS1R co-localized to sperm acrosomal domain, the site ofSLLP1 localization. The sperm nucleus was stained with DAPI. Panels: 1,phase; 2, SLLP1; 3, SAS1R; 4, merge of 2 & 3; 5, nucleus; 6, merge of 4& 5.

FIG. 8 : Inhibition of mouse fertilization by rSAS1R. Capacitated mousesperm were incubated with varying concentrations of rSAS1R prior tofertilization of cumulus intact oocytes. The percentage of fertilizedeggs decreased with increased concentration of SAS1R. SignificantP-value differences from no protein control were marked with asterisks(**, P<0.01; *, P<0.03). N=3 experiments at each concentration.

FIGS. 9A and 9B: SAS1R splice variants (SEQ ID NOs: 6, 8, 10, 19, 20 and21). Six isoforms of SAS1R were cloned from mouse ovarian cDNA libraryand aligned using ClustalW+, including the full-length protein (V1) andfive splice variants (V2-V6). The beginning of each exon is marked bythe exon number (E2-E10). The two peptides identified by surface plasmonresonance and mass spectrometric studies of SAS1R are marked withunderlined residues in variant 1. Each ovarian variant possessed a zincbinding active site signature (in boxed) and contained a putativetransmembrane domain (in shade) by specific algorithms available online(i.e., the Prediction of Transmembrane Regions and Orientation sectionof the website maintained by EMBnet and the Pasteur Institute'swebsite). The predicted signal peptide cleavage site (using toolsavailable at the website of The Center for Biological Sequence Analysisat the Technical University of Denmark) and the catalytic residue in thezinc binding active site are marked (♥ and ▾ respectively). The variants1, 4 & 6 encode a signal peptide while variants 2, 3 & 5 lack the signalpeptide (E2, exon 2). Variants 3 & 4 possess a deletion of 34 aminoacids from exon 4 and 5. Variants 5 & 6 have a replacement of exon 5with insertion of 9 residues (in italics). The GenBank accession numbersof these variants are: V1, FJ187790; V2, FJ187791; V3, FJ187792; V4,FJ187793; V5, FJ187794; V6, FJ187795.

FIGS. 10A and 10B: Alignment of deduced amino acid sequence of mouseSAS1R (V1), the human orthologue, and homologs in zebrafish andnematodes from GenBank (SEQ ID NOs:19, 23, 28 and 29), at the U.S.National Library of Medicine website maintained by the NationalInstitutes of Health. For optimal alignment, gaps (⋅) were introducedinto the sequence using GCG PileUp program (Accelrys, San Diego,Calif.). A consensus sequence was created using similarity of residuesin all four sequences (accession #: mouse, FJ187790; human,NP_001002036.3; zebrafish, NP_998800.1; nematode, NP_498405.2). In allspecies, SAS1R has a predicted signal peptide (residues in italics) anda zinc-dependent metalloprotease domain (closed rectangle). Thepredicted signal peptide cleavage site and the catalytic residue in thezinc binding active site in human sequence are marked (♥ and ▾,respectively). Mammalian orthologues contain a predicted transmembranedomain (shaded residues) while lower organisms, including chicken(accession #, XP_421101.2), zebrafish, and nematode, except Drosophila(accession #, NP_651138.1), lack this putative transmembrane domain. Theunderlined region represents the conserved zinc metalloproteasesuperfamily domain from several subfamilies (which include hatchingenzyme, astacin, astacin-like, meprin, bone morphogenesis protein 1,etc). The number of residues (aa) in each protein is shown at the end ofeach sequence.

FIGS. 11A and 11B: Oocyte Western and protease activity assay of SAS1R.FIG. 11A: Western analysis of rSAS1R probed with SAS1R preimmuneantibody (L 1) showing no immunoreactivity. Western analysis of zonafree oocytes probed with immune (L 2) and preimmune (L 3) SAS1Rantibody. The SAS1R Western profile of zona free oocytes was verysimilar to zona intact oocytes (FIG. 1A, L 5). FIG. 11B: Proteaseactivity assay of rSAS1R using fluorescent tagged synthetic peptide as asubstrate. Varying concentration of rSAS1R (multiples of 0.2 μg) wasused in 100 μl assay system for 1 h. The released fluorophore followingcleavage of the peptide bond was measured by FRET based method at anexcitation and emission of 480 and 535 nm, respectively. A concentrationdependent hydrolysis of the synthetic peptide was observed with rSAS1R(♦, correlation, R²=0.96).

FIGS. 12A to 12D: Immunofluorescent (IF) localization of SAS1R in zonafree M2 oocytes and controls. FIG. 12A: Control IF localization of SAS1Rin adult ovary section probed with preimmune antibody showing lack ofspecific signal (control of FIG. 2A). FIG. 12B: Control IF of zonaintact unpermeabilized oocytes probed with SAS1R preimmune antibodyindicating lack of specific signal (control of FIG. 2B). FIG. 12C: IFlocalization of SAS1R in zona free unpermeabilized M2 oocytes probedwith anti-SAS1R antibody. DAPI (blue) stained areas indicate M2 arrestedeccentrically positioned nuclei opposite to the microvillar domaindecorated with SAS1R expression. FIG. 12D: Control zona freeunpermeabilized oocytes stained with preimmune antibody showed noimmunoreactivity. Panels: left, phase; middle, fluorescence; right,merge image.

FIGS. 13A to 13C: Control SAS1R expression in transfected CHO-K1 cells.The cells were transfected with pcDNA3.1-TOPO vector without any SAS1Rconstruct and probed with SAS1R specific antibodies. FIG. 13A: Fixed andpermeabilized cells probed with SAS1R antibody showed no IF signal(control of FIG. 3A). FIG. 13B: Fixed and unpermeabilized CHO-K1 cellsprobed with SAS1R antibody showed no IF signal (control of FIG. 3B).FIG. 13C: Fixed and permeabilized cells probed with anti-V5 monoclonalantibody revealed no IF signal (control of FIG. 3C). Panels: left—phase;right—immunofluorescence image.

FIG. 14 : Yeast two hybrid analyses of SAS1R-SLLP1 interaction. Yeaststrain AH109 cells were grown on low stringency (LS) and high stringency(HS) plates containing X-gal transformed with both SLLP1 and SAS1Rconstructs. Growth on LS plate devoid of tryptophan and leucine confirmstransfection by both constructs. Growth on HS plate devoid oftryptophan, leucine, adenine, and histidine along with formation of bluecolonies reveals interaction between given protein constructs. Theplates were examined after three days of transfection. (A) Positiveinteraction between SV40 large T-antigen and its partner p53, as apositive control. (B) Negative interaction between large T-antigen andhuman Lamin C, as a negative control. (C) Negative interaction betweenSLLP1 and SAS1R full length. (D) Positive interaction between SLLP1 andSAS1R N-terminus. (E) Positive interaction between SLLP1 and SAS1RC-terminus.

FIG. 15 : Autoradiogram of in vitro translated SLLP1 and SAS1Rconstructs used in co-immunoprecipitation analyses (FIGS. 6A and 6B).Each construct was translated in presence of ³⁵S-methionine using rabbitreticulocyte lysate and resolved by SDS-PAGE. In 50 μl reactions, thefollowing proteins were synthesized: ND, no protein control; LF,luciferase translation control; T, large T-antigen; 53, p53 partnerprotein of T-antigen; S-SLLP1; F-SAS1R full length; N-SAS1R N-terminus;C-SAS1R C-terminus. Each translated protein contained a major band thatresolved at the expected size on SDS-PAGE analysis.

FIG. 16 : Effect of SAS1R antibody on mouse in-vitro fertilization.Cumulus intact mouse oocytes were incubated with either preimmune (lightshade) or immune (black shade) sera at 1/10 & 1/20 (N=4) or 1/80 (N=2)dilutions for 45 min followed by insemination with capacitated sperm.The number of two cell embryos was scored as fertilized eggs. Thestatistical significance between the preimmune and immune sera wascalculated by t test assuming equal variances; P<0.01.

FIG. 17 , comprising left and right panels, depicts the results ofisologous immunization of female mice and Mouse SAS1R isologous antibodyresponse. As an oocyte specific, sperm oolemmal receptor, SAS1R washypothesized herein to be a candidate contraceptive vaccinogen andimmunogen. Immunogenicity of recombinant mouse SAS1R was tested infemale mice which showed serum titers by ELISA against the recombinanttarget up to a 1/10,000 dilution after the 3rd, 4th & 5th injections(FIG. 17 , left panel). The immunoreactivity of the sera were alsostudied by immunolocalization in live mouse eggs (FIG. 17 , rightpanel). The iso-antibodies from female mice stained the microvillardomain of mouse eggs exactly as noted earlier with allo-antibodiesraised in guinea pigs (right panel). This finding confirmed thatrecombinant mouse SAS1R retained sufficient refolded epitopes to evokeiso-antibodies that cross-reacted with native SAS1R on the microvillardomain.

FIG. 18 : Day 0—Neonatal mouse ovary on day of birth stained withpre-immune [PI, left panel] or guinea pig anti-SAS1R antibodies [IM,right panel] at identical concentrations [diluted 1:500].

FIG. 19 : Day 1.5—Mouse ovary sections 1.5 days after birth stained withpre-immune [PI, left panel] or guinea pig anti-SAS1R antibodies [IM,right panel] at identical concentrations [1:500].

FIG. 20 : Day 4—Mouse ovary sections 4 days after birth stained withpre-immune [PI, left panel] or guinea pig anti-SAS1R antibodies [IM,right panel] at identical concentrations [1:500].

FIG. 21 : Day 7—Mouse ovary sections 7 days after birth stained withpre-immune [PI, left panel] or guinea pig anti-SAS1R antibodies [IM,right panel] at identical concentrations [1:500].

FIG. 22 : Day 14—Mouse ovary sections 14 days after birth [pre-pubertal]stained with pre-immune [PI, left panel] or guinea pig anti-SAS1Rantibodies [IM, right panel] at identical concentrations [1:500].

FIG. 23 : Day 28—Mouse ovary sections 28 days after birth stained withpre-immune [PI, left panel] or guinea pig anti-SAS1R antibodies [IM,right panel] at identical concentrations [1:500]. Immunoreactivity,indicating the presence of SAS1R, was observed only in oocytes.

FIG. 24 : Day 56—Adult mouse ovary sections 56 days after birth stainedwith pre-immune [PI, left panel] or guinea pig anti-SAS1R antibodies[IM, right panel] at identical concentrations [1:500].

FIG. 25 : The insert shows Western analysis of the capacitated (CP) andacrosome reacted (AR) mouse cauda epididymal sperm probed with preimmune(Pi) and immune (Im) serum. The acrosome reacted sperm clearlydemonstrated the retention of some mSLLP1, a ˜14 kD band (arrow head) inthe acrosome reacted population.

DETAILED DESCRIPTION Abbreviations and Acronyms

-   a.a.—amino acid(s)-   BSA—bovine serum albumin-   Co-IP—co-immunoprecipitation-   FITC—fluorescein isothiocyanate-   FRET—fluorescence resonance energy transfer-   FW—Far Western-   GV—germinal vesicle-   h—human (also hour)-   HPLC—reversed-phase high-pressure liquid chromatography-   HS—high stringency-   I—induced or immune-   IF—Indirect immunofluorescent-   IP—immunoprecipitation-   IPTG—Isopropyl-β-D-thiogalactopyranoside-   LB—Luria broth-   LS—low stringency-   IM—immune-   m—mouse-   MET—mouse egg-specific TolA (referred to in the provisional    application as a Colcin-like uptake protein or Colicin uptake    protein)-   min—minute-   NGS—normal goat serum-   OL—overlay-   P—purified-   PI—pre-immune-   PBS—phosphate-buffered saline-   PBST—phosphate buffered saline with 0.05% Tween 20-   PVA—polyvinylalcohol-   rec—recombinant (rec is used interchangeably with “r”)-   SAS1R—Sperm Acrosomal SLLP1 Receptor (previously referred to as ZEP)-   rSAS1R—recombinant SAS1R-   sec—second(s)-   SLLP—sperm lysozyme-like protein-   SPR—surface plasmon resonance-   U—uninduced-   ZEP—zinc endopeptidase (referred to in the provisional application    as zinc peptidase, or ZP; used interchangeably with SAS1R)-   ZFE—zona free egg-   ZIE—zona intact egg

Summary of SEQ ID NOs: Used and the Matching Names SEQ ID NOs:

SEQ ID NO:1—mouse (“m”) MET normal nucleic acid sequenceSEQ ID NO:2—mouse MET normal amino acid sequenceSEQ ID NO:3—mouse MET variant nucleic acid sequenceSEQ ID NO:4—mouse MET variant amino acid sequenceSEQ ID NO:5—mouse SAS1R Variant 2 Normal nucleic acid sequence (formerlycalled ZEP-Normal)SEQ ID NO:6—mouse SAS1R Variant 2 Normal amino acid sequence (formerlycalled ZEP-Normal)SEQ ID NO:7—mouse SAS1R Variant 5 nucleic acid sequence (formerly calledZEP Variant 1)SEQ ID NO:8—mouse SAS1R Variant 5 amino acid sequence (formerly calledZEP Variant 1)SEQ ID NO:9—mouse SAS1R Variant 3 nucleic acid sequence (formerly calledZEP Variant 2)SEQ ID NO:10—mouse SAS1R Variant 3 amino acid sequence (formerly calledZEP Variant 2)SEQ ID NO:11—mouse SLLP1 nucleic acid sequenceSEQ ID NO:12—mouse SLLP1 amino acid sequenceSEQ ID NO:13—human (“h”) SLLP1 nucleic acid sequenceSEQ ID NO:14—human SLLP1 amino acid sequenceSEQ ID NO:15—mouse SLLP2 nucleic acid sequenceSEQ ID NO:16—mouse SLLP2 mature protein amino acid sequenceSEQ ID NO:17—human SLLP2 nucleic acid sequenceSEQ ID NO:18—human SLLP2 amino acid sequenceSEQ ID NO:19—mouse SAS1R Variant 1 amino acid sequenceSEQ ID NO:20—mouse SAS1R Variant 4 amino acid sequenceSEQ ID NO:21—mouse SAS1R Variant 6 amino acid sequenceSEQ ID NO:22—human SAS1R nucleic acid sequence (GenBank accession no.NM_001002036, 1296 bp mRNA)SEQ ID NO:23—human SAS1R amino acid sequence (GenBank accession no.NP_001002036.3, 431 amino acids)SEQ ID NO:24—HELMHVLGFWH (motif in SAS1R with histidine residues for Zncoordination and conserved catalytic residue, E [glutamic acid], formspart of the catalytic pocket along with a tyrosine zinc ligand embeddedin the motif SVMHY (SEQ ID NO:25).SEQ ID NO:25—SVMHY (motif in SAS1R associated with the catalytic pocket)SEQ ID NO:26—HEXXHXXGXXH (the consensus motif of SEQ ID NO:24 can haveresidues which can be substituted with any amino acid, as indicated by“X”, that does not ablate the function of that motif).SEQ ID NO:27—SXMHY (the consensus motif of SEQ ID NO:25 can haveresidues which can be substituted with any amino acid, as indicated by“X”, that does not ablate the function of that motif).SEQ ID NOs:1-18 are the same sequences as SEQ ID NOs:1-18 ofinternational patent application WO 2006/091535 (PCT/US2006/005970;Mandal et al.; published Aug. 31, 2006), in which SAS1R was referred toas ZEP.

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. In one aspect, the term “about” meansplus or minus 20% of the numerical value of the number with which it isbeing used. Therefore, about 50% means in the range of 45%-55%.Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”

The terms “additional therapeutically active compound” or “additionaltherapeutic agent”, as used in the context of the present invention,refers to the use or administration of a compound for an additionaltherapeutic use for a particular injury, disease, or disorder beingtreated. Such a compound, for example, could include one being used totreat an unrelated disease or disorder, or a disease or disorder whichmay not be responsive to the primary treatment for the injury, diseaseor disorder being treated.

As used herein, the term “adjuvant” refers to a substance that elicitsan enhanced immune response when used in combination with a specificantigen.

As use herein, the terms “administration of” and or “administering” acompound should be understood to mean providing a compound of theinvention or a prodrug of a compound of the invention to a subject inneed of treatment.

As used herein, the term “aerosol” refers to suspension in the air. Inparticular, aerosol refers to the particlization or atomization of aformulation of the invention and its suspension in the air.

As used herein, an “agonist” is a composition of matter which, whenadministered to a mammal such as a human, enhances or extends abiological activity attributable to the level or presence of a targetcompound or molecule of interest in the mammal.

An “antagonist” is a composition of matter which when administered to amammal such as a human, inhibits a biological activity attributable tothe level or presence of a compound or molecule of interest in themammal.

As used herein, “alleviating a disease or disorder symptom,” meansreducing the severity of the symptom or the frequency with which such asymptom is experienced by a patient, or both.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino- and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

As used herein, an “analog” of a chemical compound is a compound that,by way of example, resembles another in structure but is not necessarilyan isomer (e.g., 5-fluorouracil is an analog of thymine).

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein, or chemical moiety isused to immunize a host animal, numerous regions of the antigen mayinduce the production of antibodies that bind specifically to a givenregion or three-dimensional structure on the protein; these regions orstructures are referred to as antigenic determinants. An antigenicdeterminant may compete with the intact antigen (i.e., the “immunogen”used to elicit the immune response) for binding to an antibody.

The term “antimicrobial agents” as used herein refers to anynaturally-occurring, synthetic, or semi-synthetic compound orcomposition or mixture thereof, which is safe for human or animal use aspracticed in the methods of this invention, and is effective in killingor substantially inhibiting the growth of microbes. “Antimicrobial” asused herein, includes antibacterial, antifungal, and antiviral agents.

As used herein, the term “antisense oligonucleotide” or antisensenucleic acid means a nucleic acid polymer, at least a portion of whichis complementary to a nucleic acid which is present in a normal cell orin an affected cell. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences. The antisense oligonucleotides of the invention include, butare not limited to, phosphorothioate oligonucleotides and othermodifications of oligonucleotides.

An “aptamer” is a compound that is selected in vitro to bindpreferentially to another compound (for example, the identified proteinsherein). Often, aptamers are nucleic acids or peptides because randomsequences can be readily generated from nucleotides or amino acids (bothnaturally occurring or synthetically made) in large numbers but ofcourse they need not be limited to these.

The term “binding” refers to the adherence of molecules to one another,such as, but not limited to, enzymes to substrates, ligands toreceptors, antibodies to antigens, DNA binding domains of proteins toDNA, and DNA or RNA strands to complementary strands.

“Binding partner,” as used herein, refers to a molecule capable ofbinding to another molecule.

The term “biocompatible”, as used herein, refers to a material that doesnot elicit a substantial detrimental response in the host.

As used herein, the term “biologically active fragments” or “bioactivefragment” of the polypeptides encompasses natural or synthetic portionsof the full-length protein that are capable of specific binding to theirnatural ligand or of performing the function of the protein.

The term “biological sample,” as used herein, refers to samples obtainedfrom a subject, including, but not limited to, skin, hair, tissue,blood, plasma, cells, sweat and urine.

“C19” and “C23” are names which are also used for “SLLP1” and SLLP2”,

As used herein, the term “carrier molecule” refers to any molecule thatis chemically conjugated to the antigen of interest that enables animmune response resulting in antibodies specific to the native antigen.

As used herein, the term “chemically conjugated,” or “conjugatingchemically” refers to linking the antigen to the carrier molecule. Thislinking can occur on the genetic level using recombinant technology,wherein a hybrid protein may be produced containing the amino acidsequences, or portions thereof, of both the antigen and the carriermolecule. This hybrid protein is produced by an oligonucleotide sequenceencoding both the antigen and the carrier molecule, or portions thereof.This linking also includes covalent bonds created between the antigenand the carrier protein using other chemical reactions, such as, but notlimited to glutaraldehyde reactions. Covalent bonds may also be createdusing a third molecule bridging the antigen to the carrier molecule.These cross-linkers are able to react with groups, such as but notlimited to, primary amines, sulfhydryls, carbonyls, carbohydrates, orcarboxylic acids, on the antigen and the carrier molecule. Chemicalconjugation also includes non-covalent linkage between the antigen andthe carrier molecule.

A “coding region” of a gene consists of the nucleotide residues of thecoding strand of the gene and the nucleotides of the non-coding strandof the gene which are homologous with or complementary to, respectively,the coding region of an mRNA molecule which is produced by transcriptionof the gene.

The term “competitive sequence” refers to a peptide or a modification,fragment, derivative, or homolog thereof that competes with anotherpeptide for its cognate binding site.

“Complementary” as used herein refers to the broad concept of subunitsequence complementarity between two nucleic acids, e.g., two DNAmolecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).Thus, it is known that an adenine residue of a first nucleic acid regionis capable of forming specific hydrogen bonds (“base pairing”) with aresidue of a second nucleic acid region which is antiparallel to thefirst region if the residue is thymine or uracil. Similarly, it is knownthat a cytosine residue of a first nucleic acid strand is capable ofbase pairing with a residue of a second nucleic acid strand which isantiparallel to the first strand if the residue is guanine. A firstregion of a nucleic acid is complementary to a second region of the sameor a different nucleic acid if, when the two regions are arranged in anantiparallel fashion, at least one nucleotide residue of the firstregion is capable of base pairing with a residue of the second region.Preferably, the first region comprises a first portion and the secondregion comprises a second portion, whereby, when the first and secondportions are arranged in an antiparallel fashion, at least about 50%,and preferably at least about 75%, at least about 90%, or at least about95% of the nucleotide residues of the first portion are capable of basepairing with nucleotide residues in the second portion. More preferably,all nucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

A “compound,” as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above.

As used herein, the term “conservative amino acid substitution” isdefined herein as an amino acid exchange within one of the followingfive groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

-   -   Met Leu, Ile, Val, Cys

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp

“Contraceptive”, as used herein, refers to an agent, compound, or methodthat diminishes the likelihood of or prevents conception.

A “control” cell is a cell having the same cell type as a test cell. Thecontrol cell may, for example, be examined at precisely or nearly thesame time the test cell is examined. The control cell may also, forexample, be examined at a time distant from the time at which the testcell is examined, and the results of the examination of the control cellmay be recorded so that the recorded results may be compared withresults obtained by examination of a test cell.

A “test” cell is a cell being examined.

“Cytokine,” as used herein, refers to intercellular signaling molecules,the best known of which are involved in the regulation of mammaliansomatic cells. A number of families of cytokines, both growth promotingand growth inhibitory in their effects, have been characterizedincluding, for example, interleukins, interferons, and transforminggrowth factors. A number of other cytokines are known to those of skillin the art. The sources, characteristics, targets and effectoractivities of these cytokines have been described.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

The use of the word “detect” and its grammatical variants refers tomeasurement of the species without quantification, whereas use of theword “determine” or “measure” with their grammatical variants are meantto refer to measurement of the species with quantification. The terms“detect” and “identify” are used interchangeably herein.

As used herein, a “detectable marker” or a “reporter molecule” is anatom or a molecule that permits the specific detection of a compoundcomprising the marker in the presence of similar compounds without amarker. Detectable markers or reporter molecules include, e.g.,radioactive isotopes, antigenic determinants, enzymes, nucleic acidsavailable for hybridization, chromophores, fluorophores,chemiluminescent molecules, electrochemically detectable molecules, andmolecules that provide for altered fluorescence-polarization or alteredlight-scattering.

As used herein, the term “diagnosis” refers to detecting a risk orpropensity to an addictive related disease disorder. In any method ofdiagnosis exist false positives and false negatives. Any one method ofdiagnosis does not provide 100% accuracy.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties such as ligand binding, signal transduction, cell penetrationand the like. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains.

As used herein, an “effective amount” or “therapeutically effectiveamount” means an amount sufficient to produce a selected effect, such asalleviating symptoms of a disease or disorder. In the context ofadministering compounds in the form of a combination, such as multiplecompounds, the amount of each compound, when administered in combinationwith another compound(s), may be different from when that compound isadministered alone. Thus, an effective amount of a combination ofcompounds refers collectively to the combination as a whole, althoughthe actual amounts of each compound may vary. The term “more effective”means that the selected effect is alleviated to a greater extent by onetreatment relative to the second treatment to which it is beingcompared.

As used herein, the term “effector domain” refers to a domain capable ofdirectly interacting with an effector molecule, chemical, or structurein the cytoplasm which is capable of regulating a biochemical pathway.

As used herein, the phrases “egg protein” or “egg-specific protein”refer to proteins which are expressed exclusively or predominately ineggs or ovaries. The proteins need not be expressed at all stages of eggor ovarian development.

The term “elixir,” as used herein, refers in general to a clear,sweetened, alcohol-containing, usually hydroalcoholic liquid containingflavoring substances and sometimes active medicinal agents.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

An “enhancer” is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

The term “epitope” as used herein is defined as small chemical groups onthe antigen molecule that can elicit and react with an antibody. Anantigen can have one or more epitopes. Most antigens have many epitopes;i.e., they are multivalent. In general, an epitope is roughly five aminoacids or sugars in size. One skilled in the art understands thatgenerally the overall three-dimensional structure, rather than thespecific linear sequence of the molecule, is the main criterion ofantigenic specificity.

As used herein, an “essentially pure” preparation of a particularprotein or peptide is a preparation wherein at least about 95%, andpreferably at least about 99%, by weight, of the protein or peptide inthe preparation is the particular protein or peptide.

A “fragment” or “segment” is a portion of an amino acid sequence,comprising at least one amino acid, or a portion of a nucleic acidsequence comprising at least one nucleotide. The terms “fragment” and“segment” are used interchangeably herein.

As used herein, the term “fragment,” as applied to a protein or peptide,can ordinarily be at least about 3-15 amino acids in length, at leastabout 15-25 amino acids, at least about 25-50 amino acids in length, atleast about 50-75 amino acids in length, at least about 75-100 aminoacids in length, and greater than 100 amino acids in length.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 20 nucleotides in length, typically, atleast about 50 nucleotides, more typically, from about 50 to about 100nucleotides, preferably, at least about 100 to about 200 nucleotides,even more preferably, at least about 200 nucleotides to about 300nucleotides, yet even more preferably, at least about 300 to about 350,even more preferably, at least about 350 nucleotides to about 500nucleotides, yet even more preferably, at least about 500 to about 600,even more preferably, at least about 600 nucleotides to about 620nucleotides, yet even more preferably, at least about 620 to about 650,and most preferably, the nucleic acid fragment will be greater thanabout 650 nucleotides in length.

As used herein, a “functional” biological molecule is a biologicalmolecule in a form in which it exhibits a property by which it ischaracterized. A functional enzyme, for example, is one which exhibitsthe characteristic catalytic activity by which the enzyme ischaracterized.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

As used herein, “homology” is used synonymously with “identity.”

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site having theuniversal resource locator using the BLAST tool at the NCBI website.BLAST nucleotide searches can be performed with the NBLAST program(designated “blastn” at the NCBI web site), using the followingparameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3;match reward=1; expectation value 10.0; and word size=11 to obtainnucleotide sequences homologous to a nucleic acid described herein.BLAST protein searches can be performed with the XBLAST program(designated “blastn” at the NCBI web site) or the NCBI “blastp” program,using the following parameters: expectation value 10.0, BLOSUM62 scoringmatrix to obtain amino acid sequences homologous to a protein moleculedescribed herein. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (1997,Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blastcan be used to perform an iterated search which detects distantrelationships between molecules (Id.) and relationships betweenmolecules which share a common pattern. When utilizing BLAST, GappedBLAST, PSI-Blast, and PHI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementarity between the nucleic acids, stringency of the conditionsinvolved, the length of the formed hybrid, and the G:C ratio within thenucleic acids.

By the term “immunizing a subject against an antigen” is meantadministering to the subject a composition, a protein complex, a DNAencoding a protein complex, an antibody or a DNA encoding an antibody,which elicits an immune response in the subject, and, for example,provides protection to the subject against a disease caused by theantigen or which prevents the function of the antigen.

The term “immunologically active fragments thereof” will generally beunderstood in the art to refer to a fragment of a polypeptide antigencomprising at least an epitope, which means that the fragment at leastcomprises 4 contiguous amino acids from the sequence of the polypeptideantigen.

As used herein, the term “induction of apoptosis” means a process bywhich a cell is affected in such a way that it begins the process ofprogrammed cell death, which is characterized by the fragmentation ofthe cell into membrane-bound particles that are subsequently eliminatedby the process of phagocytosis.

As used herein, the term “inhaler” refers both to devices for nasal andpulmonary administration of a drug, e.g., in solution, powder and thelike. For example, the term “inhaler” is intended to encompass apropellant driven inhaler, such as is used to administer antihistaminefor acute asthma attacks, and plastic spray bottles, such as are used toadminister decongestants.

The term “inhibit,” as used herein, refers to the ability of a compound,agent, or method to reduce or impede a described function, level,activity, rate, etc., based on the context in which the term “inhibit”is used. Preferably, inhibition is by at least 10%, more preferably byat least 25%, even more preferably by at least 50%, and most preferably,the function is inhibited by at least 75%. The term “inhibit” is usedinterchangeably with “reduce” and “block.”

The term “inhibit a complex,” as used herein, refers to inhibiting theformation of a complex or interaction of two or more proteins, as wellas inhibiting the function or activity of the complex. The term alsoencompasses disrupting a formed complex. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

The term “inhibit a protein,” as used herein, refers to any method ortechnique which inhibits protein synthesis, levels, activity, orfunction, as well as methods of inhibiting the induction or stimulationof synthesis, levels, activity, or function of the protein of interest.The term also refers to any metabolic or regulatory pathway which canregulate the synthesis, levels, activity, or function of the protein ofinterest. The term includes binding with other molecules and complexformation. Therefore, the term “protein inhibitor” refers to any agentor compound, the application of which results in the inhibition ofprotein function or protein pathway function. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

The phrase “inhibit conception”, as used herein, refers to both directand indirect inhibition of conception or impregnation, regardless of themechanism. The phrase also includes reducing the rate of conception, anddoes not necessarily mean that conception is inhibited by 100%.

As used herein “injecting or applying” includes administration of acompound of the invention by any number of routes and means including,but not limited to, topical, oral, buccal, intravenous, intramuscular,intra arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the identified compound invention or be shipped togetherwith a container which contains the identified compound. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the instructional material and the compound beused cooperatively by the recipient.

By ‘interaction” between a sperm protein and an egg protein is meant theinteraction such as binding which is necessary for an event or processto occur, such as sperm-egg binding, fusion, or fertilization. In oneaspect, the “interaction” may be similar to receptor-ligand type ofbinding or interaction.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

A “ligand” is a compound that specifically binds to a target receptor.

A “receptor” is a compound that specifically binds to a ligand.

A ligand or a receptor (e.g., an antibody) “specifically binds to” or“is specifically immunoreactive with” a compound when the ligand orreceptor functions in a binding reaction which is determinative of thepresence of the compound in a sample of heterogeneous compounds. Thus,under designated assay (e.g., immunoassay) conditions, the ligand orreceptor binds preferentially to a particular compound and does not bindin a significant amount to other compounds present in the sample. Forexample, a polynucleotide specifically binds under hybridizationconditions to a compound polynucleotide comprising a complementarysequence; an antibody specifically binds under immunoassay conditions toan antigen bearing an epitope against which the antibody was raised. Avariety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

As used herein, the term “linkage” refers to a connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding, andhydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins twoother molecules either covalently or noncovalently, e.g., through ionicor hydrogen bonds or van der Waals interactions, e.g., a nucleic acidmolecule that hybridizes to one complementary sequence at the 5′ end andto another complementary sequence at the 3′ end, thus joining twonon-complementary sequences.

“Malexpression” of a gene means expression of a gene in a cell of apatient afflicted with a disease or disorder, wherein the level ofexpression (including non-expression), the portion of the geneexpressed, or the timing of the expression of the gene with regard tothe cell cycle, differs from expression of the same gene in a cell of apatient not afflicted with the disease or disorder. It is understoodthat malexpression may cause or contribute to the disease or disorder,be a symptom of the disease or disorder, or both.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene or protein of interest. Such assays includemeasuring the level of mRNA, protein levels, etc. and can be performedby assays such as northern and western blot analyses, binding assays,immunoblots, etc. The level of expression can include rates ofexpression and can be measured in terms of the actual amount of an mRNAor protein present.

The term “nasal administration” in all its grammatical forms refers toadministration of at least one compound of the invention through thenasal mucous membrane to the bloodstream for systemic delivery of atleast one compound of the invention. The advantages of nasaladministration for delivery are that it does not require injection usinga syringe and needle, it avoids necrosis that can accompanyintramuscular administration of drugs, and trans-mucosal administrationof a drug is highly amenable to self administration.

The term “nucleic acid” typically refers to large polynucleotides. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil).

As used herein, the term “nucleic acid” encompasses RNA as well assingle and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine, anduracil). Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

The term “nucleic acid construct,” as used herein, encompasses DNA andRNA sequences encoding the particular gene or gene fragment desired,whether obtained by genomic or synthetic methods.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “oligonucleotide” typically refers to short polynucleotides,generally, no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

An “oocyte” as used herein can be categorized more specifically severalways. A “naked oocyte” is defined as a female germ cell that is notsurrounded by a continuous sheet of nurse granulose cells. A “primordialoocyte” is defined as a female germ cell that is surrounded by a singlelayer of squamous nurse granulosa cells. A “primary oocyte” is definedas a female germ cell that is surrounded by a single layer of cuboidalnurse granulose cells. A “secondary oocyte” is defined as a female germcell that is surrounded by two layers of cuboidal granulose cells. A“preeantral oocyte” is defined as a female germ cell that is surroundedby three or more layers of granulose cells but without an antral space.An “antral oocyte” is defined as a female germ cell that is surroundedby three or more layers of granulosa cells and contains evidence ofantral fluid spaces.

By describing two polynucleotides as “operably linked” is meant that asingle-stranded or double-stranded nucleic acid moiety comprises the twopolynucleotides arranged within the nucleic acid moiety in such a mannerthat at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

The term “peptide” typically refers to short polypeptides.

The term “per application” as used herein refers to administration of adrug or compound to a subject.

The term “pharmaceutical composition” shall mean a compositioncomprising at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal (for example, without limitation, a human). Those of ordinaryskill in the art will understand and appreciate the techniquesappropriate for determining whether an active ingredient has a desiredefficacious outcome based upon the needs of the artisan.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate compound or derivativecan be combined and which, following the combination, can be used toadminister the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

“Pharmaceutically acceptable” means physiologically tolerable, foreither human or veterinary application.

As used herein, “pharmaceutical compositions” include formulations forhuman and veterinary use.

“Plurality” means at least two.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof.

“Synthetic peptides or polypeptides” means a non-naturally occurringpeptide or polypeptide. Synthetic peptides or polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.Various solid phase peptide synthesis methods are known to those ofskill in the art.

By “presensitization” is meant pre-administration of at least one innateimmune system stimulator prior to challenge with an agent. This issometimes referred to as induction of tolerance.

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “preventive” or “prophylactic” treatment is a treatment administeredto a subject who does not exhibit signs, or exhibits only early signs,of a disease or disorder. A prophylactic or preventative treatment isadministered for the purpose of decreasing the risk of developingpathology associated with developing the disease or disorder.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties. As used herein, the term “promoter/regulatory sequence” meansa nucleic acid sequence which is required for expression of a geneproduct operably linked to the promoter/regulator sequence. In someinstances, this sequence may be the core promoter sequence and in otherinstances, this sequence may also include an enhancer sequence and otherregulatory elements which are required for expression of the geneproduct. The promoter/regulatory sequence may, for example, be one whichexpresses the gene product in a tissue specific manner.

A “constitutive” promoter is a promoter which drives expression of agene to which it is operably linked, in a constant manner in a cell. Byway of example, promoters which drive expression of cellularhousekeeping genes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

As used herein, “protecting group” with respect to a terminal aminogroup refers to a terminal amino group of a peptide, which terminalamino group is coupled with any of various amino-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, acyl protecting groups such as formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups such as benzyloxycarbonyl; andaliphatic urethane protecting groups, for example, tert-butoxycarbonylor adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides,vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitableprotecting groups.

As used herein, “protecting group” with respect to a terminal carboxygroup refers to a terminal carboxyl group of a peptide, which terminalcarboxyl group is coupled with any of various carboxyl-terminalprotecting groups. Such protecting groups include, for example,tert-butyl, benzyl or other acceptable groups linked to the terminalcarboxyl group through an ester or ether bond.

The term “protein” typically refers to large polypeptides. Conventionalnotation is used herein to portray polypeptide sequences: the left-handend of a polypeptide sequence is the amino-terminus; the right-hand endof a polypeptide sequence is the carboxyl-terminus.

The term “protein regulatory pathway”, as used herein, refers to boththe upstream regulatory pathway which regulates a protein, as well asthe downstream events which that protein regulates. Such regulationincludes, but is not limited to, transcription, translation, levels,activity, posttranslational modification, and function of the protein ofinterest, as well as the downstream events which the protein regulates.

The terms “protein pathway” and “protein regulatory pathway” are usedinterchangeably herein.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure. In particular, purified spermcell DNA refers to DNA that does not produce significant detectablelevels of non-sperm cell DNA upon PCR amplification of the purifiedsperm cell DNA and subsequent analysis of that amplified DNA. A“significant detectable level” is an amount of contaminate that would bevisible in the presented data and would need to be addressed/explainedduring analysis of the forensic evidence.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred toas a “recombinant host cell.” A gene which is expressed in a recombinanthost cell wherein the gene comprises a recombinant polynucleotide,produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

A “receptor” is a compound that specifically binds to a ligand.

A “ligand” is a compound that specifically binds to a target receptor.

A “recombinant cell” is a cell that comprises a transgene. Such a cellmay be a eukaryotic or a prokaryotic cell. Also, the transgenic cellencompasses, but is not limited to, an embryonic stem cell comprisingthe transgene, a cell obtained from a chimeric mammal derived from atransgenic embryonic stem cell where the cell comprises the transgene, acell obtained from a transgenic mammal, or fetal or placental tissuethereof, and a prokaryotic cell comprising the transgene.

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

As used herein, the term “reporter gene” means a gene, the expression ofwhich can be detected using a known method. By way of example, theEscherichia coli lacZ gene may be used as a reporter gene in a mediumbecause expression of the lacZ gene can be detected using known methodsby adding the chromogenic substrate o-nitrophenyl-β-galactoside to themedium (Gerhardt et al., eds., 1994, Methods for General and MolecularBacteriology, American Society for Microbiology, Washington, D.C., p.574).

A “sample,” as used herein, refers preferably to a biological samplefrom a subject, including, but not limited to, normal tissue samples,diseased tissue samples, biopsies, blood, saliva, feces, semen, tears,and urine. A sample can also be any other source of material obtainedfrom a subject which contains cells, tissues, or fluid of interest. Asample can also be obtained from cell or tissue culture.

“SLLP1” and SLLP2” are also referred to as “C19” and “C23”,respectively.

As used herein, the term “secondary antibody” refers to an antibody thatbinds to the constant region of another antibody (the primary antibody).

By the term “signal sequence” is meant a polynucleotide sequence whichencodes a peptide that directs the path a polypeptide takes within acell, i.e., it directs the cellular processing of a polypeptide in acell, including, but not limited to, eventual secretion of a polypeptidefrom a cell. A signal sequence is a sequence of amino acids which aretypically, but not exclusively, found at the amino terminus of apolypeptide which targets the synthesis of the polypeptide to theendoplasmic reticulum. In some instances, the signal peptide isproteolytically removed from the polypeptide and is thus absent from themature protein.

By “small interfering RNAs (siRNAs)” is meant, inter alia, an isolateddsRNA molecule comprised of both a sense and an anti-sense strand. Inone aspect, it is greater than 10 nucleotides in length. siRNA alsorefers to a single transcript which has both the sense and complementaryantisense sequences from the target gene, e.g., a hairpin. siRNA furtherincludes any form of dsRNA (proteolytically cleaved products of largerdsRNA, partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA) as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides.

As used herein, the term “solid support” relates to a solvent insolublesubstrate that is capable of forming linkages (preferably covalentbonds) with various compounds. The support can be either biological innature, such as, without limitation, a cell or bacteriophage particle,or synthetic, such as, without limitation, an acrylamide derivative,agarose, cellulose, nylon, silica, or magnetized particles.

By the term “specifically binds to”, as used herein, is meant when acompound or ligand functions in a binding reaction or assay conditionswhich is determinative of the presence of the compound in a sample ofheterogeneous compounds.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered and used for comparing results when administeringa test compound, or it can be a standard parameter or function which ismeasured to obtain a control value when measuring an effect of an agentor compound on a parameter or function. Standard can also refer to an“internal standard”, such as an agent or compound which is added atknown amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured. Internal standards are often a purified marker of interestwhich has been labeled, such as with a radioactive isotope, allowing itto be distinguished from an endogenous marker.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

As used herein, a “subject in need thereof” is a patient, animal,mammal, or human, who will benefit from the method of this invention.

As used herein, a “substantially homologous amino acid sequences”includes those amino acid sequences which have at least about 95%homology, preferably at least about 96% homology, more preferably atleast about 97% homology, even more preferably at least about 98%homology, and most preferably at least about 99% or more homology to anamino acid sequence of a reference antibody chain. Amino acid sequencesimilarity or identity can be computed by using the BLASTP and TBLASTNprograms which employ the BLAST (basic local alignment search tool)2.0.14 algorithm. The default settings used for these programs aresuitable for identifying substantially similar amino acid sequences forpurposes of the present invention.

“Substantially homologous nucleic acid sequence” means a nucleic acidsequence corresponding to a reference nucleic acid sequence wherein thecorresponding sequence encodes a peptide having substantially the samestructure and function as the peptide encoded by the reference nucleicacid sequence; e.g., where only changes in amino acids not significantlyaffecting the peptide function occur. Preferably, the substantiallyidentical nucleic acid sequence encodes the peptide encoded by thereference nucleic acid sequence. The percentage of identity between thesubstantially similar nucleic acid sequence and the reference nucleicacid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more.Substantial identity of nucleic acid sequences can be determined bycomparing the sequence identity of two sequences, for example byphysical/chemical methods (i.e., hybridization) or by sequence alignmentvia computer algorithm. Suitable nucleic acid hybridization conditionsto determine if a nucleotide sequence is substantially similar to areference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2× standard saline citrate(SSC), 0.1% SDS at 50° C.; preferably in 7% (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C.; preferably 7%SDS, 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDSat 50° C.; and more preferably in 7% SDS, 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.1×SSC, 0.1% SDS at 65° C. Suitable computeralgorithms to determine substantial similarity between two nucleic acidsequences include, GCS program package (Devereux et al., 1984 Nucl.Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al.,1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al., J.Mol. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res.25:3389-3402). The default settings provided with these programs aresuitable for determining substantial similarity of nucleic acidsequences for purposes of the present invention.

The term “substantially pure” describes a compound, e.g., a protein orpolypeptide which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides by columnchromatography, gel electrophoresis, or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

As used herein, the term “transgene” means an exogenous nucleic acidsequence comprising a nucleic acid which encodes a promoter/regulatorysequence operably linked to nucleic acid which encodes an amino acidsequence, which exogenous nucleic acid is encoded by a transgenicmammal.

As used herein, the term “transgenic mammal” means a mammal, the germcells of which comprise an exogenous nucleic acid.

As used herein, a “transgenic cell” is any cell that comprises a nucleicacid sequence that has been introduced into the cell in a manner thatallows expression of a gene encoded by the introduced nucleic acidsequence.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a patient or subject or administeringan agent or compound to reduce the frequency with which symptoms areexperienced.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease.

By the term “vaccine,” as used herein, is meant a composition which wheninoculated into a subject has the effect of stimulating an immuneresponse in the subject, which serves to fully or partially protect thesubject against a condition, disease or its symptoms. In one aspect, thecondition is conception. The term vaccine encompasses prophylactic aswell as therapeutic vaccines. A combination vaccine is one whichcombines two or more vaccines, or two or more compounds or agents.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer or delivery of nucleicacid to cells, such as, for example, polylysine compounds, liposomes,and the like. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,recombinant viral vectors, and the like. Examples of non-viral vectorsinclude, but are not limited to, liposomes, polyamine derivatives of DNAand the like.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

EMBODIMENTS

The present invention provides compositions and methods to prevent orinhibit SAS1R function or activity, including the use of SAS1R proteinsand fragments and homologs thereof. The present invention furtherprovides compositions and methods utilizing SAS1R, and fragments andhomologs thereof, to elicit an immune response against SAS1R. In oneaspect, administration of SAS1R or antigenic fragments or homologsthereof results in inhibiting SAS1R. In one aspect, such an immunogenicresponse and resulting inhibition of SAS1R results in a decrease infertility.

The following are useful mammalian SAS1R sequences:

mouse SAS1R Variant 1, 435 residues (SEQ ID NO: 19)MGIMGSLWPWILTMLSLLGLSMGAPSASRCSGVCS TSVPEGFTPEGSPVFQDKDIPAINQGLISEETPESSFLVEGDIIRPSPFRLLSVTNNKWPKGVGGFVEIP FLLSRKYDELSRRVIMDAFAEFERFTCIRFVAYHGQRDFVSILPMAGCFSGVGRSGGMQVVSLAPTCLRK GRGIVLHELMHVLGFWHEHSRADRDRYIQVNWNEILPGFEINFIKSRSTNMLVPYDYSSVMHYGRFAFSW RGQPTIIPLWTSSVHIGQRWNLSTSDITRVCRLYNCSRSVPDSHGRGFEAQSDGSSLTPASISRLQRLLE ALSEESGSSAPSGSRTGGQSIAGLGNSQQGWEHPPQSTFSVGALARPPQMLADASKSGPGAGADSLSLEQ FQLAQAPTVPLALFPEARDKPAPIQDAFERLAPLPGGCAPGSHIREVPRD mouse SAS1R Variant 2 (formerly called ZEP-N), 414 a.a.residues (SEQ ID NO: 6) MGAPSASRCSGVCSTSVPEGFTPEGSPVFQDKDIPAINQGLISEETPESSFLVEGDIIRPSPFRLLSVTN NKWPKGVGGFVEIPFLLSRKYDELSRRVIMDAFAEFERFTCIRFVAYHGQRDFVSILPMAGCFSGVGRSG GMQVVSLAPTCLRKGRGIVLHELMHVLGFWHEHSRADRDRYIQVNWNEILPGFEINFIKSRSTNMLVPYD YSSVMHYGRFAFSWRGQPTIIPLWTSSVHIGQRWNLSTSDITRVCRLYNCSRSVPDSHGRGFEAQSDGSS LTPASISRLQRLLEALSEESGSSAPSGSRTGGQSIAGLGNSQQGWEHPPQSTFSVGALARPPQMLADASK SGPGAGADSLSLEQFQLAQAPTVPLALFPEARDKPAPIQDAFERLAPLPGGCAPGSHIREVPRD mouse SAS1R Variant 3 (formerly calledZEP-Variant 2), 380 a.a. residues (SEQ ID NO: 10)MGAPSASRCSGVCSTSVPEGFTPEGSPVFQDKDIP AINQGLISEETPESSFLLSRKYDELSRRVIMDAFAEFERFTCIRFVAYHGQRDFVSILPMAGCFSGVGRS GGMQVVSLAPTCLRKGRGIVLHELMHVLGFWHEHSRADRDRYIQVNWNEILPGFEINFIKSRSTNMLVPY DYSSVMHYGRFAFSWRGQPTIIPLWTSSVHIGQRWNLSTSDITRVCRLYNCSRSVPDSHGRGFEAQSDGS SLTPASISRLQRLLEALSEESGSSAPSGSRTGGQSIAGLGNSQQGWEHPPQSTFSVGALARPPQMLADAS KSGPGAGADSLSLEQFQLAQAPTVPLALFPEARDKPAPIQDAFERLAPLPGGCAPGSHIREVPRD mouse SAS1R Variant 4, 401 a.a. residues(SEO ID NO: 20) MGIMGSLWPWILTMLSLLGLSMGAPSASRCSGVCSTSVPEGFTPEGSPVFQDKDIPAINQGLISEETPES SFLLSRKYDELSRRVIMDAFAEFERFTCIRFVAYHGQRDFVSILPMAGCFSGVGRSGGMQVVSLAPTCLR KGRGIVLHELMHVLGFWHEHSRADRDRYIQVNWNEILPGFEINFIKSRSTNMLVPYDYSSVMHYGRFAFS WRGQPTIIPLWTSSVHIGQRWNLSTSDITRVCRLYNCSRSVPDSHGRGFEAQSDGSSLTPASISRLQRLL EALSEESGSSAPSGSRTGGQSIAGLGNSQQGWEHPPQSTFSVGALARPPQMLADASKSGPGAGADSLSLE QFQLAQAPTVPLALFPEARDKPAPIQDAFERLAPLPGGCAPGSHIREVPRD mouse SAS1R Variant 5 (formerly called ZEP Variant 1),392 a.a. residues (SEQ ID NO: 8) MGAPSASRCSGVCSTSVPEGFTPEGSPVFQDKDIPAINQGLISEETPESSFLVEGDIIRPGVSHGVSFPD ELSRRVIMDAFAEFERFTCIRFVAYHGQRDFVSILPMAGCFSGVGRSGGMQVVSLAPTCLRKGRGIVLHE LMHVLGFWHEHSRADRDRYIQVNWNEILPGFEINFIKSRSTNMLVPYDYSSVMHYGRFAFSWRGQPTIIP LWTSSVHIGQRWNLSTSDITRVCRLYNCSRSVPDSHGRGFEAQSDGSSLTPASISRLQRLLEALSEESGS SAPSGSRTGGQSIAGLGNSQQGWEHPPQSTFSVGALARPPQMLADASKSGPGAGADSLSLEQFQLAQAPT VPLALFPEARDKPAPIQDAFERLAPLPGGCAPGSHIREVPRD mouse SAS1R Variant 6, 413 a.a. residues (SEQ ID NO: 21)MGIMGSLWPWILTMLSLLGLSMGAPSASRCSGVCS TSVPEGFTPEGSPVFQDKDIPAINQGLISEETPESSFLVEGDIIRPGVSHGVSFPNELSRRVIMDAFAEF ERFTCIRFVAYHGQRDFVSILPMAGCFSGVGRSGGMQVVSLAPTCLRKGRGIVLHELMHVLGFWHEHSRA DRDRYIQVNWNEILPGFEINFIKSRSTNMLVPYDYSSVMHYGRFAFSWRGQPTIIPLWTSSVHIGQRWNL STSDITRVCRLYNCSRSVPDSHGRGFEAQSDGSSLTPASISRLQRLLEALSEESGSSAPSGSRTGGQSIA GLGNSQQGWEHPPQSTFSVGALARPPQMLADASKSGPGAGADSLSLEQFQLAQAPTVPLALFPEARDKPA PIQDAFERLAPLPGGCAPGSHIREVPRDHuman SAS1R nucleic acid sequence- GenBank accession no, NM_001002036,1296 bp mRNA (SEQ ID NO: 22) atggagggtgtagggggtctctggccttgggtgctgggtctgctctccttgccaggtgtgatcctaggag cgcccctggcctccagctgcgcaggagcctgtggtaccagcttcccagatggcctcacccctgagggaac ccaggcctccggggacaaggacattcctgcaattaaccaagggctcatcctggaagaaaccccagagagc agcttcctcatcgagggggacatcatccggccgagtcccttccgactgctgtcagcaaccagcaacaaat ggcccatgggtggtagtggtgtcgtggaggtccccttcctgctctccagcaagtacgatgagcccagccg ccaggtcatcctggaggctcttgcggagtttgaacgttccacgtgcatcaggtttgtcacctatcaggac cagagagacttcatttccatcatccccatgtatgggtgcttctcgagtgtggggcgcagtggagggatgc aggtggtctccctggcgcccacgtgtctccagaagggccggggcattgtccttcatgagctcatgcatgt gctgggcttctggcacgagcacacgcgggccgaccgggaccgctatatccgtgtcaactggaacgagatc ctgccaggctttgaaatcaacttcatcaagtctcagagcagcaacatgctgacgccctatgactactcct ctgtgatgcactatgggaggctcgccttcagccggcgtgggctgcccaccatcacaccactttgggcccc cagtgtccacatcggccagcgatggaacctgagtgcctcggacatcacccgggtcctcaaactctacggc tgcagcccaagtggccccaggccccgtgggagagggtcccatgcccacagcactggtaggagccccgctc cggcctccctatctctgcagcggcttttggaggcactgtcggcggaatccaggagccccgaccccagtgg ttccagtgcgggaggccagcccgttcctgcagggcctggggagagcccacatgggtgggagtcccctgcc ctgaaaaagctcagtgcagaggcctcggcaaggcagcctcagaccctagcttcctccccaagatcaaggc ctggagcaggtgcccccggtgttgctcaggagcagtcctggctggccggagtgtccaccaagcccacagt cccatcttcagaagcaggaatccagccagtccctgtccagggaagcccagctctgccagggggctgtgta cctagaaatcatttcaaggggatgtccgaagattaa Human SAS1R protein-431 amino acids,GenBank accession no. NP_001002036.3 (SEQ ID NO: 23)MEGVGGLWPWVLGLLSLPGVILGAPLASSCAGACG TSFPDGLTPEGTQASGDKDIPAINQGLILEETPESSFLIEGDIIRPSPFRLLSATSNKWPMGGSGVVEVP FLLSSKYDEPSRQVILEALAEFERSTCIRFVTYQDQRDFISIIPMYGCFSSVGRSGGMQVVSLAPTCLQK GRGIVLHELMHVLGFWHEHTRADRDRYIRVNWNEILPGFEINFIKSQSSNMLTPYDYSSVMHYGRLAFSR RGLPTITPLWAPSVHIGQRWNLSASDITRVLKLYGCSPSGPRPRGRGSHAHSTGRSPAPASLSLQRLLEA LSAESRSPDPSGSSAGGQPVPAGPGESPHGWESPALKKLSAEASARQPQTLASSPRSRPGAGAPGVAQEQ SWLAGVSTKPTVPSSEAGIQPVPVQGSPALPGGCVPRNHFKGMSED

In one embodiment, the invention provides a pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and at least one eggprotein, or a homolog, fragment or derivative thereof, wherein saidprotein is capable of inducing an immune response useful for inhibitingconception in a subject. In one aspect, the invention provides apharmaceutical composition, wherein said egg protein or peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs:6, 8, 10, 19, 20, 21, and 23, and fragments and homologsthereof. In another aspect, the invention provides a pharmaceuticalcomposition further comprising at least one additional egg protein. Inone aspect, the additional egg protein is capable of inducing an immuneresponse useful for contraception.

The SAS1R protein, and fragments and homologs thereof, when administeredto a subject can inhibit fertilization or conception directly orindirectly. It can inhibit fertilization directly by acting as acompetitive inhibitor/binding agent that interacts with SLLP protein onsperm, which in turns prevents SLLP from interacting with native SAS1Ron an egg. It can inhibit fertilization or conception indirectly byacting as an immunogen that elicits an immune response against nativeSAS1R on an egg, wherein the resultant antibodies bind to the nativeSAS1R and inhibit interaction of SLLP with SAS1R or block SAS1R activityand therefore inhibits fertilization or conception.

In one embodiment, the invention provides a pharmaceutical composition,wherein said pharmaceutical composition comprises at least two differentproteins, or homologs, fragments, or derivatives thereof, wherein atleast one of said at least two different proteins comprise an amino acidsequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 19,20, 21, and 23, and fragments and homologs thereof.

In one embodiment, at least one isolated nucleic acid comprising anucleic acid sequence encoding an egg protein is administered. In oneaspect, the egg protein comprises a sequence selected from the groupconsisting of SEQ ID NOs: 6, 8, 10, 19, 20, 21, and 23, and fragmentsand homologs thereof.

An administered protein or a protein expressed by an administeredisolated nucleic acid comprising a sequence encoding the protein can actto inhibit SLLP1 and SAS1R interaction or binding.

The present invention also provides for administering at least on SLLP1protein or biologically active homologs and fragments thereof capable ofbinding with or interacting with SAS1R and preventing or inhibitingsperm and egg binding and fertilization.

It will be appreciated, of course, that the proteins or peptides of theinvention may incorporate amino acid residues which are modified withoutaffecting activity. For example, the termini may be derivatized toinclude blocking groups, i.e. chemical substituents suitable to protectand/or stabilize the N- and C-termini from “undesirable degradation”, aterm meant to encompass any type of enzymatic, chemical or biochemicalbreakdown of the compound at its termini which is likely to affect thefunction of the compound, i.e. sequential degradation of the compound ata terminal end thereof.

Blocking groups include protecting groups conventionally used in the artof peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking groups, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acid analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

Other modifications can also be incorporated without adversely affectingthe activity and these include, but are not limited to, substitution ofone or more of the amino acids in the natural L-isomeric form with aminoacids in the D-isomeric form. Thus, the peptide may include one or moreD-amino acid resides, or may comprise amino acids which are all in theD-form. Retro-inverso forms of peptides in accordance with the presentinvention are also contemplated, for example, inverted peptides in whichall amino acids are substituted with D-amino acid forms.

Acid addition salts of the present invention are also contemplated asfunctional equivalents. Thus, a peptide in accordance with the presentinvention treated with an inorganic acid such as hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organicacid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic,malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie,mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicyclicand the like, to provide a water soluble salt of the peptide is suitablefor use in the invention.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

Nucleic acids useful in the present invention include, by way of exampleand not limitation, oligonucleotides and polynucleotides such asantisense DNAs and/or RNAs; ribozymes; DNA for gene therapy; viralfragments including viral DNA and/or RNA; DNA and/or RNA chimeras; mRNA;plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structuralforms of DNA including single-stranded DNA, double-stranded DNA,supercoiled DNA and/or triple-helical DNA; Z-DNA; and the like. Thenucleic acids may be prepared by any conventional means typically usedto prepare nucleic acids in large quantity. For example, DNAs and RNAsmay be chemically synthesized using commercially available reagents andsynthesizers by methods that are well-known in the art (see, e.g., Gait,1985, OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH (IRL Press,Oxford, England)). RNAs may be produce in high yield via in vitrotranscription using plasmids such as SP65 (Promega Corporation, Madison,Wis.).

Antibodies and their Preparation

Antibodies directed against proteins, polypeptides, or peptide fragmentsthereof of the invention may be generated using methods that are wellknown in the art. For instance, U.S. patent application Ser. No.07/481,491, which is incorporated by reference herein in its entirety,discloses methods of raising antibodies to peptides. For the productionof antibodies, various host animals, including but not limited torabbits, mice, and rats, can be immunized by injection with apolypeptide or peptide fragment thereof. To increase the immunologicalresponse, various adjuvants may be used depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

In one embodiment, antibodies, or antisera, directed against SAS1R or ahomolog or fragment thereof, are useful for blocking the activity ofSAS1R, including its ability to interact with sperm.

Fragments of SAS1R may be generated and antibodies prepared against thefragments. Assays are provided herein to determine whether an antibodydirected against SAS1R or a fragment thereof have the ability to inhibitSAS1R activity or function. The assays include measuring the ability ofSAS1R to bind with or interact with SLLP proteins, as well as theability of an antibody to block SAS1R's role in fertilization. Forexample, in vitro fertilization assays are described herein using anantibody directed SAS1R and this type of assay can be used to test theability of new antibodies to block SAS1R's function. These same assayscan be used to test any compound or agent's ability to disrupt SAS1R'sinteraction with a SLLP protein or to inhibit fertilization. Proteaseassays for measuring SAS1R protease activity are also available whenneeded to confirm that a fragment or homolog of SAS1R maintains the sameactivity as the parent SAS1R molecule.

Various methods of preparing fragments of SAS1R and making antibodiesagainst SAS1R are available and these methods can be used to map thevarious regions of SAS1R that are susceptible to inhibition by anantibody.

For example, fragments of SAS1R can be prepared for use as an antigen,such as wherein the antibody binds to one of more fragments comprisingamino acids 1-25, 26-50, 51-75, 76-100, 101-125, 126-150, 151-175,176-200, 201-225, 226-250, 251-275, 276-300, 301-325, 326-350, 351-375,376-400, and 401-414 of SAS1R Variant 2 (SEQ ID NO:6) or wherein theantibody binds to one or more fragments comprising amino acids 1-25,26-50, 51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201-225,226-250, 251-275, 276-300, 301-325, 326-350, 351-375, 376-400, 401-425,and 426-435 of SAS1R Variant 1 (SEQ ID NO:19) or wherein the antibodybinds to amino acids 1-25, 26-50, 51-75, 76-100, 101-125, 126-150,151-175, 176-200, 201-225, 226-250, 251-275, 276-300, 301-325, 326-350,351-375, 376-400, 401-425, and 426-431 of SAS1R Variant 1 (SEQ IDNO:23). Such techniques can also be applied to the full-length protein.

Of course, these fragments can also be prepared to yield overlappingsequences and longer and shorter fragments can be prepared. For example,as described herein, interaction experiments between SLLP1 and SAS1Rindicate there are at least two binding regions between the two proteinswhen they interact, which may have different functions. There, fragmentsencompassing sections of the more N-terminal region of SAS1R or the moreC-terminal region of SAS1R can be prepared, such as wherein the antibodybinds to amino acids about 1 to about 121 (N-terminal) or an antibodywhich binds to about 204 to about 414 (more C-terminal) of SAS1R (SEQ IDNO:6) or an antibody which binds to similar regions of SEQ ID NO:23(human SAS1R).

The antigenic fragments of the proteins of the invention may includepeptide antigens that are at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150 or up to about 200 amino acids in length.Also included are full-length unprocessed protein as well as matureprocessed protein. These various length antigenic fragments may bedesigned in tandem order of linear amino acid sequence of the immunogenof choice, such as SAS1R, or staggered in linear sequence of theprotein. In addition, antibodies to three-dimensional epitopes, i.e.,non-linear epitopes, can also be prepared, based on, e.g.,crystallographic data of proteins. Hosts may also be injected withpeptides of different lengths encompassing a desired target sequence.Antibodies obtained from that injection may be screened against theshort antigens of SAS1R and against mature SAS1R. Antibodies preparedagainst a SAS1R peptide may be tested for activity against that peptideas well as the full length SAS1R protein. Antibodies may have affinitiesof at least about 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻¹⁰M, 10⁻¹¹M, or 10⁻¹²Mtoward the SAS1R peptide and/or the full-length SAS1R protein.

In one embodiment, the invention provides a contraceptive vaccinecomprising a pharmaceutical composition of the invention, saidcomposition comprising one or more proteins, or variants, homologs, orfragments thereof, comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 6, 8, 10, 19, 20, 21, and 23, andfragments and homologs thereof, and optionally at least one other eggprotein, or a variant, fragment, or homolog thereof.

In one embodiment, the invention provides a method for inhibitingconception in a subject, said method comprising administering to saidsubject a pharmaceutical composition comprising apharmaceutically-acceptable carrier and at least one egg protein, or ahomolog, fragment or derivative thereof, wherein said protein is capableof inducing an immune response useful for inhibiting conception in asubject. In one aspect, the egg protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 6, 8, 10, 19, 20, 21,and 23, and fragments and homologs thereof.

For the preparation of monoclonal antibodies, any technique whichprovides for the production of antibody molecules by continuous celllines in culture may be utilized. For example, the hybridoma techniqueoriginally developed by Kohler and Milstein (1975, Nature 256:495-497),the trioma technique, the human B-cell hybridoma technique (Kozbor etal., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96) may be employed to produce human monoclonal antibodies.In another embodiment, monoclonal antibodies are produced in germ-freeanimals.

In accordance with the invention, human antibodies may be used andobtained by utilizing human hybridomas (Cote et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells withEBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Furthermore, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al.,1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) bysplicing the genes from a mouse antibody molecule specific for epitopesof SLLP polypeptides together with genes from a human antibody moleculeof appropriate biological activity can be employed; such antibodies arewithin the scope of the present invention. Once specific monoclonalantibodies have been developed, the preparation of mutants and variantsthereof by conventional techniques is also available.

In one embodiment, techniques described for the production ofsingle-chain antibodies (U.S. Pat. No. 4,946,778, incorporated byreference herein in its entirety) are adapted to produceprotein-specific single-chain antibodies. In another embodiment, thetechniques described for the construction of Fab expression libraries(Huse et al., 1989, Science 246:1275-1281) are utilized to allow rapidand easy identification of monoclonal Fab fragments possessing thedesired specificity for specific antigens, proteins, derivatives, oranalogs of the invention.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′)₂ fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment; the Fab fragments which can be generated bytreating the antibody molecule with papain and a reducing agent; and Fvfragments.

The generation of polyclonal antibodies is accomplished by inoculatingthe desired animal with the antigen and isolating antibodies whichspecifically bind the antigen therefrom.

Monoclonal antibodies directed against full length or peptide fragmentsof a protein or peptide may be prepared using any well known monoclonalantibody preparation procedures, such as those described, for example,in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold SpringHarbor, N.Y.) and in Tuszynski et al. (1988, Blood, 72:109-115).Quantities of the desired peptide may also be synthesized using chemicalsynthesis technology. Alternatively, DNA encoding the desired peptidemay be cloned and expressed from an appropriate promoter sequence incells suitable for the generation of large quantities of peptide.Monoclonal antibodies directed against the peptide are generated frommice immunized with the peptide using standard procedures as referencedherein.

A nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. in Immunol. 12(3,4):125-168) and thereferences cited therein. Further, the antibody of the invention may be“humanized” using the technology described in Wright et al., (supra) andin the references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77(4):755-759).

To generate a phage antibody library, a cDNA library is first obtainedfrom mRNA which is isolated from cells, e.g., the hybridoma, whichexpress the desired protein to be expressed on the phage surface, e.g.,the desired antibody. cDNA copies of the mRNA are produced using reversetranscriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor, N.Y.).

Bacteriophage which encode the desired antibody, may be engineered suchthat the protein is displayed on the surface thereof in such a mannerthat it is available for binding to its corresponding binding protein,e.g., the antigen against which the antibody is directed. Thus, whenbacteriophage which express a specific antibody are incubated in thepresence of a cell which expresses the corresponding antigen, thebacteriophage will bind to the cell. Bacteriophage which do not expressthe antibody will not bind to the cell. Such panning techniques are wellknown in the art.

Processes such as those described above, have been developed for theproduction of human antibodies using M13 bacteriophage display (Burtonet al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNA library isgenerated from mRNA obtained from a population of antibody-producingcells. The mRNA encodes rearranged immunoglobulin genes and thus, thecDNA encodes the same. Amplified cDNA is cloned into M13 expressionvectors creating a library of phage which express human Fab fragments ontheir surface. Phage which display the antibody of interest are selectedby antigen binding and are propagated in bacteria to produce solublehuman Fab immunoglobulin. Thus, in contrast to conventional monoclonalantibody synthesis, this procedure immortalizes DNA encoding humanimmunoglobulin rather than cells which express human immunoglobulin.

The procedures just presented describe the generation of phage whichencode the Fab portion of an antibody molecule. However, the inventionshould not be construed to be limited solely to the generation of phageencoding Fab antibodies. Rather, phage which encode single chainantibodies (scFv/phage antibody libraries) are also included in theinvention. Fab molecules comprise the entire Ig light chain, that is,they comprise both the variable and constant region of the light chain,but include only the variable region and first constant region domain(CHi) of the heavy chain. Single chain antibody molecules comprise asingle chain of protein comprising the Ig Fv fragment. An Ig Fv fragmentincludes only the variable regions of the heavy and light chains of theantibody, having no constant region contained therein. Phage librariescomprising scFv DNA may be generated following the procedures describedin Marks et al., 1991, J. Mol. Biol. 222:581-597. Panning of phage sogenerated for the isolation of a desired antibody is conducted in amanner similar to that described for phage libraries comprising Fab DNA.

The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.1995, J. Mol. Biol. 248:97-105).

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). Antibodies generated in accordancewith the present invention may include, but are not limited to,polyclonal, monoclonal, chimeric (i.e., “humanized”), and single chain(recombinant) antibodies, Fab fragments, and fragments produced by a Fabexpression library.

The peptides of the present invention may be readily prepared bystandard, well-established techniques, such as solid-phase peptidesynthesis (SPPS) as described by Stewart et al. in Solid Phase PeptideSynthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.;and as described by Bodanszky and Bodanszky in The Practice of PeptideSynthesis, 1984, Springer-Verlag, New York. At the outset, a suitablyprotected amino acid residue is attached through its carboxyl group to aderivatized, insoluble polymeric support, such as cross-linkedpolystyrene or polyamide resin. “Suitably protected” refers to thepresence of protecting groups on both the α-amino group of the aminoacid, and on any side chain functional groups. Side chain protectinggroups are generally stable to the solvents, reagents and reactionconditions used throughout the synthesis, and are removable underconditions that will not affect the final peptide product. Stepwisesynthesis of the oligopeptide is carried out by the removal of theN-protecting group from the initial amino acid, and couple thereto ofthe carboxyl end of the next amino acid in the sequence of the desiredpeptide. This amino acid is also suitably protected. The carboxyl of theincoming amino acid can be activated to react with the N-terminus of thesupport-bound amino acid by formation into a reactive group such asformation into a carbodiimide, a symmetric acid anhydride or an “activeester” group such as hydroxybenzotriazole or pentafluorophenly esters.

Examples of solid phase peptide synthesis methods include the BOC methodthat utilized tert-butyloxcarbonyl as the α-amino protecting group, andthe FMOC method which utilizes 9-fluorenylmethyloxcarbonyl to protectthe α-amino of the amino acid residues, both methods of which arewell-known by those of skill in the art.

To ensure that the proteins or peptides obtained from either chemical orbiological synthetic techniques is the desired peptide, analysis of thepeptide composition should be conducted. Such amino acid compositionanalysis may be conducted using high resolution mass spectrometry todetermine the molecular weight of the peptide. Alternatively, oradditionally, the amino acid content of the peptide can be confirmed byhydrolyzing the peptide in aqueous acid, and separating, identifying andquantifying the components of the mixture using HPLC, or an amino acidanalyzer. Protein sequenators, which sequentially degrade the peptideand identify the amino acids in order, may also be used to determinedefinitely the sequence of the peptide.

Prior to its use, the peptide can be purified to remove contaminants. Inthis regard, it will be appreciated that the peptide will be purified tomeet the standards set out by the appropriate regulatory agencies. Anyone of a number of a conventional purification procedures may be used toattain the required level of purity including, for example,reversed-phase high-pressure liquid chromatography (HPLC) using analkylated silica column such as C₄-, C₈- or C₁₈-silica. A gradientmobile phase of increasing organic content is generally used to achievepurification, for example, acetonitrile in an aqueous buffer, usuallycontaining a small amount of trifluoroacetic acid. Ion-exchangechromatography can be also used to separate peptides based on theircharge.

Substantially pure peptide obtained as described herein may be purifiedby following known procedures for protein purification, wherein animmunological, enzymatic or other assay is used to monitor purificationat each stage in the procedure. Protein purification methods are wellknown in the art, and are described, for example in Deutscher et al.(ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich,San Diego).

Aptamers

The present invention is also directed to useful aptamers for blockingSAS1R function and activity, and its expression levels. In oneembodiment, an aptamer is a compound that is selected in vitro to bindpreferentially to another compound (in this case the identifiedproteins). In one aspect, aptamers are nucleic acids or peptides,because random sequences can be readily generated from nucleotides oramino acids (both naturally occurring or synthetically made) in largenumbers but of course they need not be limited to these. In anotheraspect, the nucleic acid aptamers are short strands of DNA that bindprotein targets. In one aspect, the aptamers are oligonucleotideaptamers. Oligonucleotide aptamers are oligonucleotides which can bindto a specific protein sequence of interest. A general method ofidentifying aptamers is to start with partially degenerateoligonucleotides, and then simultaneously screen the many thousands ofoligonucleotides for the ability to bind to a desired protein. The boundoligonucleotide can be eluted from the protein and sequenced to identifythe specific recognition sequence. Transfer of large amounts of achemically stabilized aptamer into cells can result in specific bindingto a polypeptide of interest, thereby blocking its function. [Forexample, see the following publications describing in vitro selection ofaptamers: Klug et al., Mol. Biol. Reports 20:97-107 (1994); Wallis etal., Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol. 4:427-429(1994); Lato et al., Chem. Biol. 2:291-303 (1995); Conrad et al., Mol.Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin. Struct. Biol.6:281-287 (1996)].

The present invention further encompasses the use of phylomers whichinhibit or prevent SAS1R function or levels.

Aptamers offer advantages over other oligonucleotide-based approachesthat artificially interfere with target gene function due to theirability to bind protein products of these genes with high affinity andspecificity. However, RNA aptamers can be limited in their ability totarget intracellular proteins since even nuclease-resistant aptamers donot efficiently enter the intracellular compartments. Moreover, attemptsat expressing RNA aptamers within mammalian cells through vector-basedapproaches have been hampered by the presence of additional flankingsequences in expressed RNA aptamers, which may alter their functionalconformation.

The idea of using single-stranded nucleic acids (DNA and RNA aptamers)to target protein molecules is based on the ability of short sequences(20 mers to 80 mers) to fold into unique 3D conformations that enablethem to bind targeted proteins with high affinity and specificity. RNAaptamers have been expressed successfully inside eukaryotic cells, suchas yeast and multicellular organisms, and have been shown to haveinhibitory effects on their targeted proteins in the cellularenvironment.

Methods of Identifying Antagonists and Inhibitors of SAS1R

As used herein, an antagonist or inhibiting agent may comprise, withoutlimitation, a drug, a small molecule, an antibody, an antigen bindingportion thereof or a biosynthetic antibody binding site that binds aparticular target protein; an antisense molecule that hybridizes in vivoto a nucleic acid encoding a target protein or a regulatory elementassociated therewith, or a ribozyme, aptamer, or small molecule thatbinds to and/or inhibits a target protein, or that binds to and/orinhibits, reduces or otherwise modulates expression of nucleic acidencoding a target protein.

This invention encompasses methods of screening compounds to identifythose compounds that act as agonists (stimulate) or antagonists(inhibit) of the protein interactions and pathways described herein.Screening assays for antagonist compound candidates are designed toidentify compounds that bind or complex with the peptides describedherein, or otherwise interfere with the interaction of the peptides withother cellular proteins. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

SAS1R assays also include those described in detail herein, such asfar-western, co-immunoprecipitation, immunoassays,immunocytochemical/immunolocalization, interaction with SLLP protein,fertilization, contraception, and immunogenicity.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,high-throughput assays, immunoassays, and cell-based assays, which arewell characterized in the art.

All assays for antagonists are common in that they call for contactingthe compound or drug candidate with a peptide identified herein underconditions and for a time sufficient to allow these two components tointeract.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, one of the peptides of the complexes described herein, orthe test compound or drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.Non-covalent attachment generally is accomplished by coating the solidsurface with a solution of the peptide and drying. Alternatively, animmobilized antibody, e.g., a monoclonal antibody, specific for thepeptide to be immobilized can be used to anchor it to a solid surface.The assay is performed by adding the non-immobilized component, whichmay be labeled by a detectable label, to the immobilized component,e.g., the coated surface containing the anchored component. When thereaction is complete, the non-reacted components are removed, e.g., bywashing, and complexes anchored on the solid surface are detected. Whenthe originally non-immobilized component carries a detectable label, thedetection of label immobilized on the surface indicates that complexingoccurred. Where the originally non-immobilized component does not carrya label, complexing can be detected, for example, by using a labeledantibody specifically binding the immobilized complex.

If the candidate compound interacts with, but does not bind to aparticular peptide identified herein, its interaction with that peptidecan be assayed by methods well known for detecting protein-proteininteractions. Such assays include traditional approaches, such as, e.g.,cross-linking, co-immunoprecipitation, and co-purification throughgradients or chromatographic columns. In addition, protein-proteininteractions can be monitored by using a yeast-based genetic systemdescribed by Fields and co-workers (Fields and Song, Nature (London),340:245-246 (1989); Chien et al., Proc. Natl. Acad. Sci. USA,88:9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl.Acad. Sci. USA, 89: 5789-5793 (1991). Complete kits for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique are available. This system can also be extended tomap protein domains involved in specific protein interactions as well asto pinpoint amino acid residues that are crucial for these interactions.

Compounds that interfere with the interaction of a peptide identifiedherein and other intra- or extracellular components can be tested asfollows: usually a reaction mixture is prepared containing the productof the gene and the intra- or extracellular component under conditionsand for a time allowing for the interaction and binding of the twoproducts. To test the ability of a candidate compound to inhibitbinding, the reaction is run in the absence and in the presence of thetest compound. In addition, a placebo may be added to a third reactionmixture, to serve as positive control. The binding (complex formation)between the test compound and the intra- or extracellular componentpresent in the mixture is monitored as described hereinabove. Theformation of a complex in the control reaction(s) but not in thereaction mixture containing the test compound indicates that the testcompound interferes with the interaction of the test compound and itsreaction partner.

To assay for antagonists, the peptide may be added to a cell along withthe compound to be screened for a particular activity and the ability ofthe compound to inhibit the activity of interest in the presence of thepeptide indicates that the compound is an antagonist to the peptide. Thepeptide can be labeled, such as by radioactivity.

Other assays and libraries are encompassed within the invention, such asthe use of Phylomers® and reverse yeast two-hybrid assays (see Watt,2006, Nature Biotechnology, 24:177; Watt, U.S. Pat. No. 6,994,982; Watt,U.S. Pat. Pub. No. 2005/0287580; Watt, U.S. Pat. No. 6,510,495; Barr etal., 2004, J. Biol. Chem., 279:41:43178-43189; the contents of each ofthese publications is hereby incorporated by reference herein in theirentirety). Phylomers® are derived from sub domains of natural proteins,which makes them potentially more stable than conventional short randompeptides. Phylomers® are sourced from biological genomes that are nothuman in origin. This feature significantly enhances the potencyassociated with Phylomers® against human protein targets. Phylogica'scurrent Phylomer® library has a complexity of 50 million clones, whichis comparable with the numerical complexity of random peptide orantibody Fab fragment libraries. An Interacting Peptide Library,consisting of 63 million peptides fused to the B42 activation domain,can be used to isolate peptides capable of binding to a target proteinin a forward yeast two hybrid screen. The second is a Blocking PeptideLibrary made up of over 2 million peptides that can be used to screenfor peptides capable of disrupting a specific protein interaction usingthe reverse two-hybrid system.

The Phylomer® library consists of protein fragments, which have beensourced from a diverse range of bacterial genomes. The libraries arehighly enriched for stable subdomains (15-50 amino acids long). Thistechnology can be integrated with high throughput screening techniquessuch as phage display and reverse yeast two-hybrid traps.

The present application discloses compositions and methods forinhibiting the proteins described herein, and those not disclosed whichare known in the art are encompassed within the invention. For example,various modulators/effectors are known, e.g. antibodies, biologicallyactive nucleic acids, such as antisense molecules, RNAi molecules, orribozymes, aptamers, peptides or low-molecular weight organic compoundsrecognizing said polynucleotides or polypeptides.

The present invention also encompasses pharmaceutical and therapeuticcompositions comprising the compounds of the present invention.

The present invention is also directed to pharmaceutical compositionscomprising the compounds of the present invention. More particularly,such compounds can be formulated as pharmaceutical compositions usingstandard pharmaceutically acceptable carriers, fillers, solubilizingagents and stabilizers known to those skilled in the art.

Vaccines and Immunogens

In one embodiment, the invention relates to methods and reagents forimmunizing and treating a subject with an antigenic compound of theinvention such as SAS1R and fragments and homologs thereof, to elicitspecific cellular and humoral immune-responses against such specificantigens. The invention provides methods of using specifically preparedimmunogen in fresh or lyophilized liposome, proper routes ofadministration of the immunogen, proper doses of the immunogen, andspecific combinations of heterologous immunization including DNA primingin one administration route followed by liposome-mediated proteinantigen boost in a different route to tailor the immune responses inrespects of enhancing cell mediated immune response, cytokine secretion,humoral immune response, especially skewing T helper responses to be Th1or a balanced Th1 and Th2 type. For more detail, see Klinefelter (U.S.patent application Ser. No. 11/572,453, which claims priority tointernational patent application PCT/US2005/026102).

A homolog herein is understood to comprise an immunogenic polypeptidehaving at least 70%, preferably at least 80%, more preferably at least90%, still more preferably at least 95%, still more preferably at least98% and most preferably at least 99% amino acid sequence identity withthe naturally occurring SAS1R polypeptides mentioned above and is stillcapable of eliciting at least the immune response obtainable thereby. Ahomolog or analog may herein comprise substitutions, insertions,deletions, additional N- or C-terminal amino acids, and/or additionalchemical moieties, such as carbohydrates, to increase stability,solubility, and immunogenicity.

In one embodiment of the invention, the present immunogenic polypeptidesas defined herein, are glycosylated. Without wishing to be bound by anyparticular theory, it is hypothesized herein that by glycosylation ofthese polypeptides the immunogenicity thereof may be increased.Therefore, in one embodiment, the aforementioned immunogenic polypeptideas defined herein before, is glycosylated, having a carbohydrate contentvarying from 10-80 wt %, based on the total weight of the glycoproteinor glycosylated polypeptide. More preferably said carbohydrate contentranges from 15-70 wt %, still more preferably from 20-60 wt %. Inanother embodiment, said glycosylated immunogenic polypeptide comprisesa glycosylation pattern that is similar to that of the correspondingzona pellucida glycoprotein (or fragment thereof) of the human that istreated. It is hypothesized that this even further increases theimmunogenicity of said polypeptide. Thus, it is preferred that theimmunogenic polypeptide comprises a glycosylation pattern that issimilar to that of the corresponding SAS1R glycoprotein.

In one embodiment, the source of a polypeptide comprises an effectiveamount of an immunogenic polypeptide selected from SAS1R protein, andimmunologically active homologs thereof and fragments thereof, or anucleic acid sequence encoding said immunogenic polypeptide.

In one embodiment, the present method of immunization comprises theadministration of a source of immunogenically active polypeptidefragments, said polypeptide fragments being selected from SAS1R proteinfragments and/or homologs thereof as defined herein before, saidpolypeptide fragments comprising dominant CTL and/or HTL epitopes andwhich fragments are between 18 and 45 amino acids in length. Peptideshaving a length between 18 and 45 amino acids have been observed toprovide superior immunogenic properties as is described in WO 02/070006.

Peptides may advantageously be chemically synthesized and may optionallybe (partially) overlapping and/or may also be ligated to othermolecules, peptides, or proteins. Peptides may also be fused to formsynthetic proteins, as in Welters et al. (Vaccine. 2004 Dec. 2;23(3):305-11). It may also be advantageous to add to the amino- orcarboxy-terminus of the peptide chemical moieties or additional(modified or D-) amino acids in order to increase the stability and/ordecrease the biodegradability of the peptide. To improve immunogenicity,immuno-stimulating moieties may be attached, e.g. by lipidation orglycosylation. To enhance the solubility of the peptide, addition ofcharged or polar amino acids may be used, in order to enhance solubilityand increase stability in vivo.

For immunization purposes, the aforementioned immunogenic polypeptidesof the invention may also be fused with proteins, such as, but notlimited to, tetanus toxin/toxoid, diphtheria toxin/toxoid or othercarrier molecules. The polypeptides according to the invention may alsobe advantageously fused to heatshock proteins, such as recombinantendogenous (murine) gp96 (GRP94) as a carrier for immunodominantpeptides as described in (references: Rapp U K and Kaufmann S H, IntImmunol. 2004 April; 16(4):597-605; Zugel U, Infect Immun. 2001 June;69(6):4164-7) or fusion proteins with Hsp70 (Triebel et al; WO9954464).

The individual amino acid residues of the present immunogenic(poly)peptides of the invention can be incorporated in the peptide by apeptide bond or peptide bond mimetic. A peptide bond mimetic of theinvention includes peptide backbone modifications well known to thoseskilled in the art. Such modifications include modifications of theamide nitrogen, the alpha carbon, amide carbonyl, complete replacementof the amide bond, extensions, deletions, or backbone cross-links. See,generally, Spatola, Chemistry and Biochemistry of Amino Acids, Peptidesand Proteins, Vol. VII (Weinstein ed., 1983). Several peptide backbonemodifications are known and can be used in the practice of theinvention.

Amino acid mimetics may also be incorporated in the polypeptides. An“amino acid mimetic” as used here is a moiety other than a naturallyoccurring amino acid that conformationally and functionally serves as asubstitute for an amino acid in a polypeptide of the present invention.Such a moiety serves as a substitute for an amino acid residue if itdoes not interfere with the ability of the peptide to elicit an immuneresponse against the native SAS1R T cell epitopes. Amino acid mimeticsmay include non-protein amino acids. A number of suitable amino acidmimetics are known to the skilled artisan, they includecyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine,adamantylacetic acid and the like. Peptide mimetics suitable forpeptides of the present invention are discussed by Morgan and Gainor,(1989) Ann. Repts. Med. Chem. 24:243-252.

In one embodiment, the present method comprises the administration of acomposition comprising one or more of the present immunogenicpolypeptides as defined herein above, and at least one excipient.Excipients are well known in the art of pharmacy and may for instance befound in textbooks such as Remington's pharmaceutical sciences, MackPublishing, 1995.

The present method for immunization may further comprise theadministration, and in one aspect, the co-administration, of at leastone adjuvant. Adjuvants may comprise any adjuvant known in the art ofvaccination and may be selected using textbooks like Current Protocolsin Immunology, Wiley Interscience, 2004.

Adjuvants are herein intended to include any substance or compound that,when used, in combination with an antigen, to immunize a human or ananimal, stimulates the immune system, thereby provoking, enhancing orfacilitating the immune response against the antigen, preferably withoutgenerating a specific immune response to the adjuvant itself. In oneaspect, adjuvants can enhance the immune response against a givenantigen by at least a factor of 1.5, 2, 2.5, 5, 10, or 20, as comparedto the immune response generated against the antigen under the sameconditions but in the absence of the adjuvant. Tests for determining thestatistical average enhancement of the immune response against a givenantigen as produced by an adjuvant in a group of animals or humans overa corresponding control group are available in the art. The adjuvantpreferably is capable of enhancing the immune response against at leasttwo different antigens. The adjuvant of the invention will usually be acompound that is foreign to a human, thereby excluding immunostimulatorycompounds that are endogenous to humans, such as e.g. interleukins,interferons, and other hormones.

A number of adjuvants are well known to one of ordinary skill in theart. Suitable adjuvants include, e.g., incomplete Freund's adjuvant,alum, aluminum phosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), DDA (2 dimethyldioctadecylammoniumbromide), polyIC, Poly-A-poly-U, RIBI™., GERBU™, Pam3™, Carbopol™,Specol™, Titermax™, tetanus toxoid, diphtheria toxoid, meningococcalouter membrane proteins, diphtheria protein CRM₁₉₇. Preferred adjuvantscomprise a ligand that is recognized by a Toll-like-receptor (TLR)present on antigen presenting cells. Various ligands recognized by TLR'sare known in the art and include e.g. lipopeptides (see, e.g., WO04/110486), lipopolysaccharides, peptidoglycans, liopteichoic acids,lipoarabinomannans, lipoproteins (from mycoplasma or spirochetes),double-stranded RNA (poly I:C), unmethylated DNA, flagellin,CpG-containing DNA, and imidazoquinolines, as well derivatives of theseligands having chemical modifications.

The methods of immunization of the present application further encompassthe administration, including the co-administration, of a CD40 bindingmolecule in order to enhance a CTL response and thereby enhance thetherapeutic effects of the methods and compositions of the invention.The use of CD40 binding molecules is described in WO 99/61065,incorporated herein by reference. The CD40 binding molecule ispreferably an antibody or fragment thereof or a CD40 Ligand or a variantthereof, and may be added separately or may be comprised within acomposition according to the current invention. Such effective dosageswill depend on a variety of factors including the condition and generalstate of health of the patient. Thus, dosage regimens can be determinedand adjusted by trained medical personnel to provide the optimumtherapeutic or prophylactic effect.

In the present method, the one or more immunogenic polypeptides aretypically administered at a dosage of about 1 ug/kg patient body weightor more at least once. Often dosages are greater than 10 ug/kg.According to the present invention, the dosages preferably range from 1ug/kg to 1 mg/kg.

In one embodiment typical dosage regimens comprise administering adosage of 1-1000 ug/kg, more preferably 10-500 ug/kg, still morepreferably 10-150 ug/kg, once, twice or three times a week for a periodof one, two, three, four or five weeks. According to one embodiment,10-100 ug/kg is administered once a week for a period of one or twoweeks.

The present method, in one aspect, comprises administration of thepresent immunogenic polypeptides and compositions comprising them viathe injection, transdermal, or oral route. In another, embodiment of theinvention, the present method comprises vaginal administration of thepresent immunogenic polypeptides and compositions comprising them.

Another aspect of the invention relates to a pharmaceutical preparationcomprising as the active ingredient the present source of a polypeptideas defined herein before. More particularly pharmaceutical preparationcomprises as the active ingredient one or more of the aforementionedimmunogenic polypeptides selected from the group of SAS1R proteins,homologues thereof and fragments of said SAS1R proteins and homologsthereof, or, alternatively, a gene therapy vector as defined hereinabove.

The present invention further provides a pharmaceutical preparationcomprising one or more of the immunogenic polypeptides of the invention.The concentration of said polypeptide in the pharmaceutical compositioncan vary widely, i.e., from less than about 0.1% by weight, usuallybeing at least about 1% by weight to as much as 20% by weight or more.

The composition may comprise a pharmaceutically acceptable carrier inaddition to the active ingredient. The pharmaceutical carrier can be anycompatible, non-toxic substance suitable to deliver the immunogenicpolypeptides or gene therapy vectors to the patient. For polypeptides,sterile water, alcohol, fats, waxes, and inert solids may be used as thecarrier. Pharmaceutically acceptable adjuvants, buffering agents,dispersing agents, and the like, may also be incorporated into thepharmaceutical compositions.

In one embodiment, the present pharmaceutical composition comprises anadjuvant, as defined in more detail herein before. Adjuvants useful forincorporation in the present composition are preferably selected fromthe group of ligands that are recognized by a Toll-like-receptor (TLR)present on antigen presenting cells, including lipopeptides,lipopolysaccharides, peptidoglycans, liopteichoic acids,lipoarabinomannans, lipoproteins (from mycoplasma or spirochetes),double-stranded RNA (poly I:C), unmethylated DNA, flagellin,CpG-containing DNA, and imidazoquinolines, as well derivatives of theseligands having chemical modifications. The routineer will be able todetermine the exact amounts of anyone of these adjuvants to beincorporated in the present pharmaceutical preparations in order torender them sufficiently immunogenic. According to another preferredembodiment, the present pharmaceutical preparation may comprise one ormore additional ingredients that are used to enhance CTL immunity asexplained herein before. According to a particularly preferredembodiment the present pharmaceutical preparation comprises a CD40binding molecule.

Methods of producing pharmaceutical compositions comprising polypeptidesare described in U.S. Pat. Nos. 5,789,543 and 6,207,718. The preferredform depends on the intended mode of administration and therapeuticapplication.

In one embodiment, the present immunogenic proteins or polypeptides areadministered by injection. The parenteral route for administration ofthe polypeptide is in accordance with known methods, e.g. injection orinfusion by intravenous, intraperitoneal, intramuscular, intra-arterial,subcutaneous, or intralesional routes. The protein or polypeptide may beadministered continuously by infusion or by bolus injection. A typicalcomposition for intravenous infusion could be made up to contain 10 to50 ml of sterile 0.9% NaCl or 5% glucose optionally supplemented with a20% albumin solution and between 10 ug and 50 mg, preferably between 50ug and 10 mg, of the polypeptide. A typical pharmaceutical compositionfor intramuscular injection would be made up to contain, for example,1-10 ml of sterile buffered water and between 10 ug and 50 mg,preferably between 50 ug and 10 mg, of the polypeptide of the presentinvention. Methods for preparing parenterally administrable compositionsare well known in the art and described in more detail in varioussources, including, for example, Remington's Pharmaceutical Science(15th ed., Mack Publishing, Easton, Pa., 1980) (incorporated byreference in its entirety for all purposes).

For convenience, immune responses are often described in the presentinvention as being either “primary” or “secondary” immune responses. Aprimary immune response, which is also described as a “protective”immune response, refers to an immune response produced in an individualas a result of some initial exposure (e.g., the initial “immunization”)to a particular antigen. Such an immunization can occur, for example, asthe result of some natural exposure to the antigen (for example, frominitial infection by some pathogen that exhibits or presents theantigen). Alternatively, the immunization can occur because ofvaccinating the individual with a vaccine containing the antigen. Forexample, the vaccine can be a vaccine comprising one or more antigenicepitopes or fragments of SAS1R.

The vaccine can also be modified to express other immune activators suchas IL2, and costimulatory molecules, among others.

Another type of vaccine that can be combined with antibodies to anantigen is a vaccine prepared from a cell lysate of interest, inconjunction with an immunological adjuvant, or a mixture of lysates fromcells of interest plus DETOX™ immunological adjuvant. Vaccine treatmentcan be boosted with anti-antigen antibodies, with or without additionalchemotherapeutic treatment.

When used in vivo for therapy, the antibodies of the subject inventionare administered to the subject in therapeutically effective amounts(i.e., amounts that have desired therapeutic effect). They will normallybe administered parenterally. The dose and dosage regimen will dependupon the degree of the infection, the characteristics of the particularantibody or immunotoxin used, e.g., its therapeutic index, the patient,and the patient's history. Advantageously the antibody or immunotoxin isadministered continuously over a period of 1-2 weeks. Optionally, theadministration is made during the course of adjunct therapy such asantimicrobial treatment, or administration of tumor necrosis factor,interferon, or other cytoprotective or immunomodulatory agent.

For parenteral administration, the antibodies will be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are inherently nontoxic, and non-therapeutic. Examples of suchvehicle are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate can also be used. Liposomes can be used as carriers. The vehiclecan contain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theantibodies will typically be formulated in such vehicles atconcentrations of about 1.0 mg/ml to about 10 mg/ml.

Use of IgM antibodies can be preferred for certain applications;however, IgG molecules by being smaller can be more able than IgMmolecules to localize to certain types of infected cells.

There is evidence that complement activation in vivo leads to a varietyof biological effects, including the induction of an inflammatoryresponse and the activation of macrophages (Unanue and Benecerraf,Textbook of Immunology, 2nd Edition, Williams & Wilkins, p. 218 (1984)).The increased vasodilation accompanying inflammation can increase theability of various agents to localize. Therefore, antigen-antibodycombinations of the type specified by this invention can be used in manyways. Additionally, purified antigens (Hakomori, Ann. Rev. Immunol.2:103, 1984) or anti-idiotypic antibodies (Nepom et al., Proc. Natl.Acad. Sci. USA 81: 2864, 1985; Koprowski et al., Proc. Natl. Acad. Sci.USA 81: 216, 1984) relating to such antigens could be used to induce anactive immune response in human patients.

The antibody compositions used are formulated and dosages established ina fashion consistent with good medical practice taking into account thecondition or disorder to be treated, the condition of the individualpatient, the site of delivery of the composition, the method ofadministration, and other factors known to practitioners. The antibodycompositions are prepared for administration according to thedescription of preparation of polypeptides for administration, infra.

As is well understood in the art, biospecific capture reagents includeantibodies, binding fragments of antibodies which bind to activatedintegrin receptors on metastatic cells (e.g., single chain antibodies,Fab′ fragments, F(ab)′2 fragments, and scFv proteins and affibodies(Affibody, Teknikringen 30, floor 6, Box 700 04, Stockholm SE-10044,Sweden; See U.S. Pat. No. 5,831,012, incorporated herein by reference inits entirety and for all purposes)). Depending on intended use, theyalso can include receptors and other proteins that specifically bindanother biomolecule.

The hybrid antibodies and hybrid antibody fragments include completeantibody molecules having full length heavy and light chains, or anyfragment thereof, such as Fab, Fab′, F(ab′)2, Fd, scFv, antibody lightchains and antibody heavy chains. Chimeric antibodies which havevariable regions as described herein and constant regions from variousspecies are also suitable. See for example, U.S. Application No.20030022244.

Initially, a predetermined target object is chosen to which an antibodycan be raised. Techniques for generating monoclonal antibodies directedto target objects are well known to those skilled in the art. Examplesof such techniques include, but are not limited to, those involvingdisplay libraries, xeno or humab mice, hybridomas, and the like. Targetobjects include any substance which is capable of exhibitingantigenicity and are usually proteins or protein polysaccharides.Examples include receptors, enzymes, hormones, growth factors, peptidesand the like. It should be understood that not only are naturallyoccurring antibodies suitable for use in accordance with the presentdisclosure, but engineered antibodies and antibody fragments which aredirected to a predetermined object are also suitable.

The present application discloses compositions and methods forinhibiting the proteins described herein, and those not disclosed whichare known in the art are encompassed within the invention. For example,various modulators/effectors are known, e.g. antibodies, biologicallyactive nucleic acids, such as antisense molecules, RNAi molecules, orribozymes, aptamers, peptides or low-molecular weight organic compoundsrecognizing said polynucleotides or polypeptides, as well as the proteinitself and fragments thereof.

The present invention further encompasses the identification offunctional fragments for the use of SAS1R for use as antigens forcontraceptive antibodies as well as its use as an immunogen and as anantifertility vaccine.

In one embodiment, a mimotope analysis of full length SAS1R can beperformed by subdividing the sequence into a series of 15 amino acidpeptides, with each peptide overlapping by three amino acids. Allpeptides can be biotinylated and allowed to bind to streptavidin-coatedwells in 96-well plates. The reactivity of various antisera can bedetected by enzyme-linked immunosorbent assay (ELISA). After blockingnon-specific binding, SAS1R antibody can be added sequentially (i.e.,either affinity-purified anti-SAS1R or affinity-purifiedanti-full-length recombinant SAS1R), followed by the sequential additionof peroxidase-conjugated secondary antibody, and peroxidase substrate.

The optical density of each well can be read at 450 nm and duplicatewells averaged. The average value obtained from a similar ELISA usingcontrol serum (i.e., preimmune serum) can be subtracted from the test Igvalues and the resultant values plotted to determine which linearepitopes are recognized by the Ig.

The second and third components in the strategy to identify functionalfragments of SAS1R rely on the synthesis of non-biotinylated peptidescorresponding to the epitopes (peptides) predicted by the mimotopeanalysis. To determine whether any of the epitopes recognized bymimotope analysis are exposed on the egg, immunocytochemical stainingwith the Ig, without and with each of the peptides, can performed.

Methods for reducing fertility in females using peptides can be found,for example, in Klinefelter (U.S. patent application Ser. No.11/572,453, filed Feb. 19, 2008, based on international patentapplication PCT/US2005/026102, filed Jul. 22, 2005).

Pharmaceutical Compositions and Administration

The present invention is also directed to pharmaceutical compositionscomprising the compounds of the present invention. More particularly,such compounds can be formulated as pharmaceutical compositions usingstandard pharmaceutically acceptable carriers, fillers, solubilizingagents and stabilizers known to those skilled in the art.

The invention is also directed to methods of administering the compoundsof the invention to a subject. In one embodiment, the invention providesa method of treating a subject by administering compounds identifiedusing the methods of the invention description. Pharmaceuticalcompositions comprising the present compounds are administered to asubject in need thereof by any number of routes including, but notlimited to, topical, oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

In accordance with one embodiment, a method of treating a subject inneed of such treatment is provided. The method comprises administering apharmaceutical composition comprising at least one compound of thepresent invention to a subject in need thereof. Compounds identified bythe methods of the invention can be administered with known compounds orother medications as well.

The invention also encompasses the use of pharmaceutical compositions ofan appropriate compound, and homologs, fragments, analogs, orderivatives thereof to practice the methods of the invention, thecomposition comprising at least one appropriate compound, and homolog,fragment, analog, or derivative thereof and apharmaceutically-acceptable carrier.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 1 ng/kg/day and 100mg/kg/day.

The invention encompasses the preparation and use of pharmaceuticalcompositions comprising a compound useful for treatment of the diseasesdisclosed herein as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

It will be understood by the skilled artisan that such pharmaceuticalcompositions are generally suitable for administration to animals of allsorts. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,geese, and turkeys. The invention is also contemplated for use incontraception for nuisance animals such as rodents.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which may beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per kilogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per kilogram of body weight of the animal. Morepreferably, the dosage will vary from about 10 mg to about 1 g perkilogram of body weight of the animal.

The compound may be administered to an animal as frequently as severaltimes daily, or it may be administered less frequently, such as once aday, once a week, once every two weeks, once a month, or even leesfrequently, such as once every several months or even once a year orless. The frequency of the dose will be readily apparent to the skilledartisan and will depend upon any number of factors, such as, but notlimited to, the type and severity of the condition or disease beingtreated, the type and age of the animal, etc.

Suitable preparations of vaccines include injectables, either as liquidsolutions or suspensions, however, solid forms suitable for solution in,suspension in, liquid prior to injection, may also be prepared. Thepreparation may also be emulsified, or the polypeptides encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine preparation may also include minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and/or adjuvants which enhance the effectiveness ofthe vaccine.

The invention is also directed to methods of administering the compoundsof the invention to a subject. In one embodiment, the invention providesa method of treating a subject by administering compounds identifiedusing the methods of the invention. Pharmaceutical compositionscomprising the present compounds are administered to an individual inneed thereof by any number of routes including, but not limited to,topical, oral, intravenous, intramuscular, intra arterial,intramedullary, intrathecal, intraventricular, transdermal,subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual,or rectal means.

In accordance with one embodiment, a method of treating and vaccinatinga subject in need of such treatment is provided. The method comprisesadministering a pharmaceutical composition comprising at least onecompound of the present invention to a subject in need thereof.Compounds identified by the methods of the invention can be administeredwith known compounds or other medications as well.

For oral administration, the active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonate,and the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

A variety of vaginal drug delivery systems is known in the art. Suitablesystems include creams, foams, tablets, gels, liquid dosage forms,suppositories, and pessaries. Mucoadhesive gels and hydrogels,comprising weakly crosslinked polymers which are able to swell incontact with water and spread onto the surface of the mucosa, have beenused for vaccination with peptides and proteins through the vaginalroute previously. The present invention further provides for the use ofmicrospheres for the vaginal delivery of peptide and protein drugs. Moredetailed specifications of vaginally administered dosage forms includingexcipients and actual methods of preparing said dosage forms are known,or will be apparent, to those skilled in this art. For example,Remington's Pharmaceutical Sciences (15th ed., Mack Publishing, Easton,Pa., 1980) is referred to.

The invention also includes a kit comprising the composition of theinvention and an instructional material which describes adventitiallyadministering the composition to a cell or a tissue of a mammal. Inanother embodiment, this kit comprises a (preferably sterile) solventsuitable for dissolving or suspending the composition of the inventionprior to administering the compound to the mammal.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviation the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the peptide of the invention or be shipped together witha container which contains the peptide. Alternatively, the instructionalmaterial may be shipped separately from the container with the intentionthat the instructional material and the compound be used cooperativelyby the recipient.

Other techniques known in the art may be used in the practice of thepresent invention, including those described in international patentapplication WO 2006/091535 (PCT/US2006/005970), the entirety of which isincorporated by reference herein.

The invention is now described with reference to the following Examplesand Embodiments. Without further description, it is believed that one ofordinary skill in the art can, using the preceding description and thefollowing illustrative examples, make and utilize the present inventionand practice the claimed methods. The following working examplestherefore, are provided for the purpose of illustration only andspecifically point out the preferred embodiments of the presentinvention, and are not to be construed as limiting in any way theremainder of the disclosure. Therefore, the examples should be construedto encompass any and all variations which become evident as a result ofthe teaching provided herein.

EXAMPLES Example 1

Materials & Methods:

Identification of SAS1R by SPR: Using purified soluble rSLLP1 as bait,SAS1R was initially identified as a SLLP1 binding partner using mouseoocyte protein lysates.

Purification of mouse SAS1R and SLLP1: Mature SAS1R (without signalsequence, 414 a.a.) was cloned by PCR from mouse ovary cDNA, expressedin E. coli and purified by affinity column chromatography. SLLP1 waspurified as described earlier (2).

Antibody production and Western analyses: Antibody to purified rSAS1Rwas raised in guinea pigs. Specificity of the antibody was testedagainst rSAS1R and mouse oocyte extracts following SDS-PAGE and westernblotting using HRP-2^(nd) antibody and TMB substrate.

IF localization of SAS1R in mouse ovary: Fixed ovary sections wereprobed with anti-SAS1R antibody followed by Cy3-2^(nd) antibody andimaged with UV-microscopy.

IF microscopy of oocytes, early embryos and sperm: Zona intact-, zonafree oocytes, early embryos and sperm were collected as before (10),blocked and probed with 1^(st) and 2^(nd) antibody and imaged withUV-microscopy. For co-LC studies, gametes were incubated with solublerSAS1R or rSLLP1, washed, probed with 1^(st)- and 2^(nd) antibodies andimaged with UV-microscopy.

Scanning confocal microscopy: Mouse oocytes (GV, M2) and early embryos(2, 4, 8 cell & blastocyst) were further examined. The indirectly IFstained preparations were washed, fixed, permeabilized, treated withRNase, stained with Sytox, mounted in slow-fade and imaged with confocalmicroscopy.

Localization of SAS1R in transformed CHO-KJ Cells: A full length SAS1Rconstruct in pcDNA3.1/V5/His-TOPO vector was used to transform cells,probed with anti-SAS1R or C-terminal V5 antibody followingpermeabilization or not.

Protease Activity assay of SAS1R: Protease activity was assayed withpurified soluble rSAS1R using the EnzChek® Peptidase/Protease Assay Kit.

Far-western analysis: Purified rSAS1R on nitrocellulose membrane wasoverlaid or not with soluble rSLLP1, washed and probed with anti-SLLP1antibody.

Analysis of SLLP1-SAS1R Interactions by Y2H System 3: Mature SLLP1(c-term 128 a.a.) and SAS1R fragments (mature 414 aa without signalpeptide, N-term 121 aa and C-term 210 a.a.) were cloned in pGADT7 andpGBKT7 vectors respectively, transformed AH109 yeast cells, grown on lowand high stringency selection media and observed for expression ofreporter genes.

Co-IP of in vitro translated proteins: SLLP1 and SAS1R constructs (sameas Y2H) were in vitro translated with ³⁵S-methionine in rabbitreticulocyte lysate and co-IP with tagged monoclonal antibodies from thepartner proteins.

Gamete preparation and IVF: Epididymal sperm from sexually mature andoocytes from 6 to 8 weeks old ICR mice were used for gamete studies andIVF. Capacitated sperm in presence or absence of rSAS1R were used toinseminate cumulus intact oocytes and fertilization was scored bycounting two cell embryos.

Methods for FIGS. 9-15

Identification of SAS1R by surface plasmon resonance (SPR): Cumulus andzona free mouse oocytes (n=˜1000) were suspended in 500 μl of Dulbecco'sPBS, freeze-thawed three times over the range −80° C. to 37° C., andthen mixed by vortexing. The mixtures were centrifuged for 5 min at13000×g at room temperature, pellets were discarded and the supernatantswere passed over a BIACORE® Sensor Chip CM5 (Biacore AB, Uppsala,Sweden) containing bound soluble recombinant mouse SLLP1 (bindingconcentration used at 1 μg/μl, 200 μl) or no SLLP1 (negative control) ata flow rate of 10 μl/min at 25° C. Following sample binding, the chipswere washed in PBS and eluted in 5 μl of 0.1% trifluoroacetic acid at aflow rate of 12 μl/min at 25° C. For each SPR analysis, 7 to 10 samplesbound to the chip were collected and pooled. The pooled samples, withvolumes ranging from ˜35 to 50 μl, were digested with trypsin andanalyzed by mass spectrometry in the Biomolecular Research Facility atthe University of Virginia. Few proteins were identified as putativeligands and one in particular, SAS1R, showed EST database informaticsthat indicated specificity to the oocyte and fertilized ovum.

Expression and purification of mouse SAS1R and SLLP1: BLAST analysis ofpeptide microsequences from mass spectrometry matched a hypothetical 414amino acid mouse protein, GenBank accession NP_766127, belonging to anastacin-like protein. A mouse ovarian cDNA library (Ambion, TX) wasamplified with gene-specific forward and reverse primers containing NdeIand NotI restriction sites, respectively, obtained from Invitrogen(Carlsbad, Calif.). PCR was performed for 40 cycles in a PTC 200 DNAEngine (MJ Research, MA). PCR reaction products were separated onagarose gels, and the amplicon (˜1200 base pairs) was gel extracted bythe freeze-thaw method and cloned in a pCR2.1-TOPO vector (Invitrogen,CA). Multiple cDNA clones were sequenced in both directions usingvector-derived primers on a Perkin-Elmer Applied Biosystems DNAsequencer at the Biomolecular Research Facility, University of VirginiaHealth System. Inserts were restriction digested, gel purified andcloned into a predigested pET-28b(+) vector at the NdeI and NotIrestriction sites. This vector added nucleotides encoding twenty aminoacids including a six histidine tag at the N-terminus and ten aminoacids including a six histidine tag at the C-terminus. Ligatedconstructs were used to transform competent cells of E. coli strainBL21DE3 (Novagen, WI). A 2 L culture from a single colony was grown tooptical density of 0.6 at 600 nm at 37° C. in Luria broth (LB) media.Isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma, MO) was then added toa final concentration of 1 mM to induce expression. Following 3 h ofinduction, the bacteria were collected by centrifugation. Therecombinant protein was isolated from the insoluble fraction of the E.coli, dissolved in 8 M urea in binding buffer (20 mM Tris-HCl, pH 7.9, 5mM imidazole, and 0.5 M NaCl), and purified by chromatography on a Hisbinding Ni²⁺ chelation affinity resin column (Novagen, NJ). The purifiedprotein fractions were stored at −80° C. until used. Relative proteinconcentrations were determined by optical density at 280 nm and byCoomassie plus Bradford reagent (Pierce, Ill.). Mouse SLLP1 was purifiedas described earlier (2).

Polyclonal antibody production and Western analyses: Followingcollection of pre-immune sera by heart puncture, adult male guinea pigswere injected with 150 μg of purified recombinant SAS1R in completeFreund's adjuvant (Sigma). Booster injections were given twice atintervals of 21 days with 150 μg of recSAS1R in incomplete Freund'sadjuvant. For all immunizations, one-half of the antigen emulsion wasinjected intramuscularly in the hind legs and the other halfsubcutaneously at two super-scapular sites. Animals were exsanguinatedby heart puncture 10 days after the third immunization and blood wascollected in serum separation tubes (Becton Dickinson, Franklin Lakes,N.J.). After centrifugation at 3200×g for 10 min, the serum was removed,aliquoted, and frozen. Two adult male guinea pigs were injected with theadjuvants lacking SAS1R immunogen on an identical regimen to producecontrol sera. Specificity of the antisera was tested against recombinantSAS1R protein. Recombinant SAS1R protein (50 ng/lane) was solubilized inLaemmli buffer (2×), and proteins were resolved on a 12% SDS-PAGE(Criterion™, Bio-Rad) gel at 20 mA. Proteins were then blotted tonitrocellulose membrane (0.2 μm, Bio-Rad), and all blots were blockedwith 5% nonfat dry milk in phosphate buffered saline with 0.05% Tween 20(PBST) for 45 min at room temperature. For immunoblotting, 1:25,000 or1:50,000 dilutions of the anti-recombinant SAS1R guinea pig sera wereincubated for two hours. The blots were then washed three times for 10min in PBST, incubated with a 1:5000 dilution of peroxidase-conjugateddonkey anti-guinea pig IgG secondary antibody for 1 h, and washed twotimes for 10 min in PBST and three times for 10 min in PBS. The blotswere then developed in TMB peroxidase substrate(3,3′,5,5′-tetramethylbenzidine; KPL, MD).

This antiserum was also tested against mouse oocyte extracts following1D SDS-PAGE and western blotting. Oocytes (150/lane) from super-ovulatedmice were solubilized in Laemmli buffer and proteins were resolved on a12% SDS-PAGE gel at 20 mA. Proteins were then blotted to nitrocellulosemembranes and were blocked with 5% nonfat dry milk in PBST for 30 min atroom temperature. For immunoblotting, a 1:1,000 dilution of guinea piganti-recombinant SAS1R serum was incubated overnight. Blots were washedthree times for 10 min in PBS-T and incubated with a 1:2500 dilution ofperoxidase-conjugated goat anti-guinea pig IgG secondary antibody for 1hour. The blots were then washed twice for 10 min in PBST and threetimes for 10 min in PBS and were developed in TMB peroxidase substrate.

Indirect immunofluorescent (IF) localization of SAS1R in the mouseovary: Ovaries were collected from superovulated mice, fixed in 4%paraformaldehyde for 24 hrs, embedded in paraffin, and sectioned, andthe resulting specimens were mounted on glass slides. At five minuteintervals the slides were serially rehydrated by successive immersionsin microclear (xylene) twice, absolute ethanol twice, 95% ethanol, 80%ethanol, 70% ethanol, and double distilled water. Specimens were blockedwith 10% NGS (Normal Goat Serum) in PBS for 30 min in a humid chamberand incubated with guinea pig anti-recombinant SAS1R polyclonal antibody(1:200) in 0.1% NGS/PBS overnight at 4° C. Slides were thrice washedwith PBS for 5 min and incubated with secondary goat anti-guinea pig/Cy3Red antibody (1:200) (Jackson ImmunoResearch, PA) in 0.1% NGS/PBS for 1h at 37° C. in the dark. Slides were washed thrice for five min withPBS, mounted with slow-fade reagent (Molecular Probes, CA), coverslipssealed with nail polish, and specimens visualized under a Carl ZeissStandard 18 ultraviolet microscope. Images were captured using MrGrab1.0 (Carl Zeiss Vision GmbH, Germany).

Indirect IF microscopy of oocytes, early embryos, and sperm: The fate ofSAS1R during early development was studied by standard indirectimmunofluorescence microscopy. Metaphase II eggs and subsequentembryonic stages including 2, 4, 8 cells and blastocyst stages, wereobtained as described earlier (15) and incubated with 5% NGS/TYH mediafor 30 min. Oocytes and early embryos were incubated with guinea piganti-recombinant SAS1R polyclonal antibody (1:200) in 0.1% NGS/media for1 h at 37° C. and 5% CO₂. Preparations were washed five times andincubated with goat anti-guinea pig/Cy3 Red antibody (1:200) (JacksonImmunoResearch, PA) in 0.10% NGS/media for 1 h at 37° C. and 5% CO₂,washed again, mounted in media on glass slides, and visualized under aZeiss Standard 18 ultraviolet microscope as above.

Mouse oocytes were also examined for co-localization of SLLP1 bindingsites and SAS1R localization. Oocytes were incubated with graduallyincreasing concentrations (0-100 μg/ml) of recombinant SLLP1 for 1 h,and then oocytes were washed five times and incubated with guinea piganti-recombinant SAS1R polyclonal antibody (1:200) and ratanti-recombinant SLLP1 polyclonal antibody (1:200), simultaneously, in0.1% NGS/media for 1 h at 37° C. and 5% CO₂. Oocytes were washed fivetimes and incubated with goat anti-guinea pig/Cy3 Red antibody (1:200)(Jackson ImmunoResearch, PA) and goat anti-rat/FITC Green antibody(1:200) (Jackson ImmunoResearch, PA), in 0.10% NGS/media for 1 h at 37°C. and 5% CO₂. Oocytes were washed and mounted in media on glass slides.SLLP1 and SAS1R images were captured separately with MrGrab 1.0 (CarlZeiss Vision GmbH, Germany) on the Zeiss Standard 18 ultravioletmicroscope and were merged digitally to evaluate their respectivelocalizations.

Similarly capacitated mouse sperm were also examined for co-localizationof SAS1R binding sites and SLLP1 localization. Sperms were incubatedwith increasing concentrations (0-100 μg/ml) of rSAS1R for 1 h, thensperm were washed two times at 700 g for 8 min and droplets were made onglass slide. These droplets were air-dried for 30 min and rehydratedagain with 20 μl of PBS for 5 min. Now in the droplets, sperm were fixedwith 20 μl of 4% paraformaldehyde for 25 min and then washed with 20 μlof PBS thrice for 5 min each. Now the droplets were blocked with 10% NGSfor 30 min and incubated with guinea pig anti-rSAS1R polyclonal antibody(1:200) and rat anti-rSLLP1 polyclonal antibody (1:200) simultaneously,in 0.1% NGS/media for 1 h at 37° C. and 5% CO₂. All the droplets werewashed three times and incubated with goat anti-guinea pig/Cy3 redantibody (1:200, Jackson ImmunoResearch, PA) and goat anti-rat/FITCgreen antibody (1:200, Jackson ImmunoResearch, PA), in 0.1% NGS/mediafor 1 h at 37° C. and 5% CO₂. Sperms were washed and mounted in media onglass slides. SLLP1 and SAS1R images were captured separately withMrGrab 1.0 (Carl Zeiss Vision GmbH, Germany) on the Zeiss Standard 18ultraviolet microscope and were merged digitally to evaluate theirrespective localizations.

Scanning confocal microscopy: Mouse oocytes and early embryos were alsostudied by scanning confocal microscopy. The preparations were washedthree times in PBS containing 1% BSA (PBS/BSA) and then fixed in 4%paraformaldehyde in PBS-polyvinylalcohol (PVA) for 20 min at roomtemperature. Following fixation, oocytes and embryos were washed 5 timesin PBS/BSA and permeabilized with 0.5% Triton X-100 in PBS for 20 min atroom temperature. Specimens were washed five times in PBS/BSA, placed in0.4 mg/ml RNase (Sigma, USA) in PBS/BSA for 30 min, and stained with 20nM Sytox (Molecular Probes, CA) for 10 min. Oocytes and embryos werethen extensively washed, placed in slow-fade equilibration media(Molecular Probes, USA) for about 1 min, and then mounted on slides inslow fade mounting media. Images were obtained on a Zeiss 410 Axiovert100 Microsystems LSM confocal microscope. For each panel, 4-sec scanswere averaged four times per line using a 63× oil lens equipped with azoom factor of two. Attenuation, contrast, brightness, and pinholeaperture remained constant.

Localization of SAS1R in transformed CHO-K1 Cells: Full length SAS1R wascloned into pcDNA3.1/V5/His-TOPO vector (Invitrogen, CA) from a PCRproduct generated from a mouse ovary cDNA library (Ambion, TX) usinggene-specific forward and reverse primers. PCR reaction products wereseparated on agarose gels, and the amplicon was gel extracted byfreeze-thawing and cloned. Multiple cDNA clones were sequenced in bothdirections using vector-derived primers on a Perkin-Elmer AppliedBiosystems DNA sequencer to verify the insert sequence.

Adherent Chinese hamster ovary (CHO-K1) cells maintained in Ham's F-12nutrient medium (F12-Ham 1X; Invitrogen, CA) supplemented with fetalcalf serum (10% v/v) and 1 mM sodium pyruvate were used for alltransfection experiments. Cells were maintained at 37° C. in ahumidified 5% CO₂ atmosphere and the media changed every second day. Theday before transfection, cells were seeded on 6-well plates containingpoly-D-lysine coated 12 mm round coverslips (BD BioCoat™ Cellware,Franklin Lakes, N.J.), and were used at approximately 50-70% confluence.Cells were transfected with Lipofectamine™ 2000 (pDNA: lipofectamine; 4μg: 10p, per well) in a final volume of 500 μl of Opti-MEM (Invitrogen,CA). Cells were then incubated at 37° C. for 5 h after which the mediacontaining the transfection complex was removed. After transfection, thecells were washed in PBS, supplemented with F12 media, and incubated for48 h.

The cells were fixed briefly with 4% paraformaldehyde inphosphate-buffered saline (PBS), blocked with 10% FBS (Fetal BovineSerum) in PBS for 30 min, and a cohort permeabilized with 0.05% NP-40.Cells were incubated on the cover slips with anti-SAS1R guinea pigpolyclonal antibody for 1 h or mouse anti-V5 tag monoclonal antibody,and immune-complexes were visualized with fluorescein isothiocyanate(FITC)-conjugated donkey anti-guinea pig or mouse antibody (JacksonImmunoResearch Laboratories, PA). Cover slips were mounted with slowfade(Molecular Probes, CA), sealed with nail polish, and visualized byultraviolet microscopy as noted above.

Protease Activity assay of SAS1R: Protease activity was assayed inserial dilutions (0-2000 ng) of purified bacterial recombinant SAS1Rusing the EnzChek® Peptidase/Protease Assay Kit (Molecular Probes, OR),which provides a FRET (fluorescence resonance energy transfer)-basedmethod for quantitation of a wide range of protease activities. Thisassay was performed in 100 μl size reactions and repeated three timeswith varying concentrations of recombinant SAS1R. Fluorescence wasmeasured over 24 h following the manufacturer's protocol. Proteaseactivity of recombinant SAS1R appeared within 1 h and increased within24 h. SLLP1 was used as a negative control. The assay method wasvalidated using purified trypsin as a positive control (Sigma, MO).

Far-Western analysis between SAS1R and SLLP1: Purified recombinant SAS1Rwas resolved by SDS-PAGE and transferred to nitrocellulose membranes.The membrane was probed with c-terminal anti-his monoclonal antibody anddeveloped with TMB. Duplicate SAS1R blotted strips were overlaid withmouse rSLLP1 or not (with 5 μg/ml in blocking buffer i.e., 5% milk inPBS with 0.05% Tween 20 [PBST]) for 90 min at room temperature. Beforeprotein overlay, the strips were incubated with blocking buffer for 1 h.Following overlay, the strips were incubated with either anti-SLLP1(ADD2) monoclonal antibody for 1 h in blocking buffer. After washing inPBST (×4), the strips were probed with goat anti-mouse HRP secondaryantibody in blocking buffer for 1 h, washed in PBST (×4) followed by PBS(×1), and developed with TMB.

Analysis of SLLP1-SAS1R Interactions by Yeast Matchmaker Two-Hybrid(Y2H) System 3: Protein-protein interactions between mouse SLLP1 andmouse SAS1R were studied in the advanced GAL4 based yeast two-hybridsystem 3 (Clontech, CA). The mature, mSLLP1 protein (C-terminal 128residues) was cloned as a fusion to the GAL4 activation domain in thepGADT7 vector utilizing EcoRI and BamHI sites. Full-length mouse SAS1R(414 residues, without signal sequence), an N-terminal SAS1R protein(121 residues), and a C-terminal 210 residue protein were each fused tothe GAL4 DNA-binding domain in the pGBKT7 vector utilizing EcoRI andBamHI sites. The plasmids were amplified in TOP10 E. coli cells(Invitrogen, CA) using ampicillin (pGADT7 vector) or kanamycin (pGBKT7vector) at 50 μg/ml.

To test interactions between the constructs, strain AH109 yeast cellswere made competent using 1×TE/1×LiAc and PEG/LiAc solutions (followingthe manufacturer's protocol), transformed with single, double, or emptyconstructs, and plated on low stringency (LS) and high stringency (HS)plates. The low stringency selection plates were deficient in tryptophanand leucine, and this selection confirmed the co-transformation by bothplasmids which supplied the missing amino acids in the pGBKT7 and pGADT7vectors, respectively. The high stringency plates were deficient ofadenine and histidine in addition to tryptophan and leucine, and thiswas used for evaluation of interaction between the test plasmids. Thisyeast two-hybrid system 3 uses the expression of three reportergenes—ADE2, HIS3, and MEL1 (or LacZ)—under the control of distinct GAL4upstream activating sequences and TATA boxes. The ADE2 and HIS3reporters provide strong nutritional selections to the yeast strain,AH109. Furthermore, the expression of MEL1 gene (an endogenousGAL4-responsive gene), which encodes a secretory α-galactosidase enzyme,was used to screen and visualize the blue colonies indicative ofpositive interaction by incorporating X-α-gal directly into thestringent culture plates. The cells were grown at 30° C. for 5 to 6days. The experiments were repeated 3 times.

Co-Immunoprecipitation (co-IP) of in vitro translated proteins:Interactions between SLLP1 and SAS1R were evaluated using in vitrotranslated proteins from rabbit reticulocyte lysates labeled with³⁵S-methionine. The pGADT7 and pGBKT7 expression plasmids, which encodeN-terminal HA and N-terminal c-Myc epitopes, respectively, were employedto produce radiolabeled SLLP1 and SAS1R recombinant proteins in vitro,utilizing the Quick Coupled Transcription/Translation (TNT) system(Promega, WI). The TNT reactions were performed for 90 min at 30° C. in1.5 ml Eppendorf tubes containing the plasmid (≤2 μg), TNT reactionbuffer, T7 RNA polymerase, an amino acid mixture lacking methionine,RNasin ribonuclease inhibitor, rabbit reticulocyte lysate, and³⁵S-methionine (GE Healthcare, NJ) in 50 μl aliquots per themanufacturer's protocol. The profile of the radiolabeled proteins wasanalyzed by SDS-PAGE (15% gels) using 5 μl of TNT product along with 15μl of 2× reducing sample buffer (Pierce, Ill.). Followingelectrophoresis, the gels were fixed (50% methanol, 10% glacial aceticacid and 40% H₂O) for 30 min, equilibrated in 20% ethanol/10% glycerolfor 30 min, dried between sheets of cellophane for 1 h (GelAir dryingsystem, Bio-Rad, CA), and exposed to image intensifying phosphorscreens. The ionizing radiation profiles were imaged using aPhosphorImager (Storm 860, GE Healthcare, NJ) and analyzed by ImageQuantsoftware.

High affinity anti-c-Myc and anti-HA monoclonal antibodies coupled toagarose beads were employed to immunoprecipitate proteins (IP) and theirradiolabeled putative interacting partners (Co-IP) using the Profoundc-Myc/HA Tag IP/Co-IP system (Pierce, Ill.). For Co-IP reactions, thespecific TNT products, ranging from 7-23 μl were gently mixed and keptat 4° C. for 1 h. Simultaneously the anti-c-Myc and anti-HA antibodybeads were incubated with 5% milk in PBST (phosphate buffer saline with0.05% Tween 20) for 1 h at 4° C. in Handy spin columns (Pierce, Ill.)with gentle end-over-end mixing to prevent loss of beads during thesubsequent steps. Following blocking, the antibody beads were incubatedwith IP or Co-IP samples overnight at 4° C. in Handee spin columns withgentle end-over-end mixing. The beads were then precipitated and washed3 times in 500 μl of PBST at 4000×g for 10 sec with 3 gentle mixesduring each wash. The IP and co-IP products were then recovered from thebeads in 23 μl of 2× non-reducing sample buffer, heated at 99° C. for 5min, centrifuged, and mixed with 2 μl of β-mercaptoethanol, and 20 μl ofthis mixture was loaded per lane for SDS-PAGE analysis using Criteriongels (Bio-Rad, CA). After electrophoresis, the gels were fixed, airdried, exposed to phosphor screens, and imaged as described above foranalysis of TNT products.

Gamete preparation and in vitro fertilization: ICR mice were used in allexperiments. Suspensions of epididymal spermatozoa from sexually maturemale mice were prepared for insemination of isolated oocytes. Oocyteswere obtained from 6 to 8 weeks old females superovulated with 10 IUPMSG and 10 IU hCG, injected intraperitoneally at 48 h intervals.Females were killed 16 h after hCG injection, and both oviducts wereimmediately removed, placed in mineral oil, and flushed to recoveroocytes.

In vitro fertilization was conducted with cumulus intact oocytes usingsperm dispersed from cauda epididymides. Sperm were placed for 5 min in200 μl drops of fertilization medium under paraffin oil. The spermsuspension was diluted to a concentration of 10⁶ sperm/ml in a volume of200 μl and then incubated for 120 min in a humidified tissue cultureincubator (37° C., 5% CO₂ in air) to allow capacitation. The spermatozoawere incubated with varying concentrations (0-100 μg/ml) of purifiedrSAS1R for the last 60 min of capacitation. Cumulus masses were placedin 135 μl drops of fertilization medium (one mass per drop) underparaffin oil, and fifteen microliters of the sperm suspension (final:10⁵ sperm/ml) was added to each cumulus mass drop. Thus, recombinantprotein was present in the incubation droplet during gamete interaction.Six hours following insemination, oocytes were relocated to 100 μl dropsof fertilization medium under mineral oil. Following overnightincubation, eggs were stained in 10 μg/ml Hoechst dye for 10 min andwashed 3 times in fertilization medium. The eggs were then placed in 5μl of fertilization medium between a microscope slide and an elevatedcoverslip, and visualized at 160× using light and fluorescencemicroscopy (Zeiss Axioplan). Two-cell embryos were scored as fertilized,while one-celled oocytes were scored as unfertilized.

Isoimmunization of Female Mice:

Female mice received five injections at intramuscular andintraperitoneal sites with 40 μg of purified recombinant SAS1R incomplete Freund's adjuvant (primary immunization) or incomplete Freund'sadjuvant (booster immunizations) at weeks 1, 3, 5, 7 and 9. Sera werecollected on weeks 2, 4, 6, 8 and 10. Anti-SAS1R titers were checkedafter the 3^(rd), 4^(th) and 5^(th) bleeds by ELISA using 100 ng ofrecombinant SAS1R in 100 mM carbonate—bicarbonate buffer, pH 9.6 (Kurthet al., 2008) and by immunofluorescence microscopy on oocytes as notedabove.

Results:

Identification of SAS1R: To identify SLLP1 putative oolemmalreceptor(s), ligand affinity panning was performed with mouse egg lysateusing soluble rSLLP1 as bait using surface plasmon resonance (SPR).Peptide analysis of eluted proteins matched to several proteinsincluding an oocyte specific protein, SAS1R. Six SAS1R splice variantswere cloned with three signal sequences (FIGS. 9 and 10 ). All sixvariants contained the zinc binding active site motif, a characteristicof zinc dependent metalloproteases, and a putative transmembrane domain.Three SAS1R variants (V1, V4, and V6) revealed a putative signalsequence followed by a predicted cleavage site between residues SMG-AP.SAS1R variants revealed deletions of 34 residues from exons 4 and 5 (V3,V4) or deletion and insertion in exon 5 (V5, V6). Variants 5 and 6showed 31 a.a. deletions and 9 a.a. insertion in the 5^(th) exon.

SAS1R was found to be conserved among mammals (FIGS. 10A and 10B, 67%identity to human) and its homologs can be traced to lower invertebrates(identity, 42% zebrafish; 36% nematode). Alignment reveals conservationof signal peptide and the zinc binding signatures.

Proform sizes: V1—435-421 a.a. [G-A]; V2—414 (SEQ ID NO:6); V3—380;V4—402-381 [G-A]; V5—392; V6—413-390 [G-A].

The Zn metal ion binding catalytic pocket is underlined in FIGS. 9A and9B. The consensus motif HELMHVLGFWH (SEQ ID NO:24) with histidineresidues for Zn coordination and conserved catalytic residue, E[glutamic acid], forms part of the catalytic pocket along with atyrosine zinc ligand embedded in the motif SVMHY (SEQ ID NO:25). Theseconsensus motifs have residues which can be substituted with any aminoacid that does not ablate the activity, i.e., iEXXHXXGXXH (SEQ ID NO:26)and SXMHY (SEQ ID NO:27).

The Zn binding active site cleft in SAS1R is formed by two distinctN-terminal and C-terminal domains on either side and lined byevolutionarily conserved histidine residues. Of four highly conservedhistidines, three (H161, H165, and H171) are predicted with highconfidence to be involved in Zn coordination.

SAS1R isoforms and protease assay: The mature rSAS1R (414 residues; SEQID NO:6) was expressed in E. coli with histidine tag and purified byaffinity chromatography. The purified rSAS1R revealed two major bands(˜50 & ˜25 kD) and several minor bands (FIG. 1A). The ˜25 kD band wasfound to be a 210 residue C-terminal fragment of SAS1R by Edmandegradation (FIGS. 9A and 9B; begins at a.a. residue position 226,Variant 1). Each of the affinity purified bands identified by Coomassiestaining immunoreacted with anti-his antibody confirming them as rSAS1Rproteins. The C-terminal fragments appear to result from proteolysisduring purification.

Guinea pig anti-rSAS1R recognized the rSAS1R proteins (FIG. 1A, L4;e.g., ˜50 kD, ˜25 kD). Protein extracts from zona-intact and zona-freemouse eggs showed microheterogeneity of immunoreactive bands of nativeSAS1R between ˜51 and ˜31 kD (FIG. 1A, L5; 11, L2), with major bands of˜45 and ˜31 kD and minor bands of ˜51, ˜49 and ˜42 kD. Pre-immunecontrol antibody showed no reactivity to rSAS1R or egg extracts (FIG.11A). SAS1R microheterogeneity likely corresponds to isoforms derivedfrom the six splice variants, which are predicted to encode proteins of47.4, 45.2, 44.8, 42.5, 43.7 and 41.4 kD, while the lower ˜31 kD proteinmay be a processed form following endo-proteolytic cleavage. Thepresence of masses higher than those of deduced from primary sequencessuggest post-translational modifications of SAS1R isoforms. Theidentical SAS1R profile between zona free and zona intact oocytes,suggest that native SAS1R isoforms reside predominately with the oocyteand not with zona pellucida.

Purified rSAS1R was tested for protease activity using a fluorescentconjugated synthetic peptide substrate (FIG. 1 ). rSAS1R exhibited dosedependent proteolytic activity assayed by fluorescence resonance energytransfer (FRET) method over the range 200-2000 ng. rSAS1R demonstratedprotease activity within one hour (FIG. 11B) and the hydrolysis werelinear until 24 h (FIG. 1 ). Mouse rSLLP1 purified by identicalprocedures to rSAS1R from same E. coli strain was used as a negativecontrol. The results also indicated that E. coli expressed rSAS1Rrefolded sufficiently to retain proteolytic activity.

Oocyte specific microvillar surface expression of SAS1R: Using indirectimmunofluorescence (IF), SAS1R was localized exclusively in oocytecytoplasm (FIG. 2A, 12A). SAS1R was not localized in oocytes withinprimordial follicles but was first detected in primary follicles withmaximum staining intensity noted in secondary follicles and relativelyless in large antral follicles. This indicated that, within ovariantissues, SAS1R is specific to the oocyte. IF of live, zona-intact andzona-free ovulated M2 oocytes prior to fertilization showed that SAS1Rwas localized asymmetrically on the oocyte surface (FIG. 2B, 12C), beingconcentrated in a dome corresponding to the oolemmal microvillar region(6) which is antipodal to the eccentric nucleus. The concentrated SAS1Rstaining on the microvillar surface was similar whether the zonapellucida was intact or absent.

Parallel studies found that SAS1R is localized in live human eggsretrieved for in vitro fertilization purposes.

SAS1R confocal localization in oocyte and early embryo: In ovulated GVstage oocytes, SAS1R was observed throughout the ooplasm and wasparticularly concentrated at the oocyte periphery (FIG. 3 , GV). In M2phase oocytes, SAS1R was concentrated in the microvillar domain of theoolemma antipodal to the M2 nucleus and in the membrane of the firstpolar body. The observations imply that in the GV to M2 transition, are-orientation of membrane components including SAS1R takes place.Observations of zygotes with two pronuclei revealed that SAS1R was nolonger polarized in the microvillar domain but now showed only weakstaining of the plasma membrane (PN-II). In 2-cell through morulaestages diffuse, punctuate SAS1R staining was observed mainly in theperivitelline space (PVS), but occasionally on the oolemma. This lowlevel of diffuse, punctate staining remained mainly in the PVS throughearly blastocyst stage and then disappeared in late blastocyst stages.Thus, SAS1R demonstrated the greatest intensity of staining in theplasma membranes of GV and M2 oocytes, which suggested SAS1R plays itsmain role at the oocyte surface of pre-zygotic stages of development.Subsequently, the protein appeared to aggregate in the membrane of earlyzygote and then to be shed into the PVS.

SAS1R is a membrane protein: Full length SAS1R with a C-terminal V5 tagwas expressed in mammalian CHO-K1 cells and the protein was localized inpermeabilized and unpermeabilized transfected cells by IF. Theanti-rSAS1R antibody localized SAS1R in the cytoplasm and on the cellmembranes of permeabilized cells and at the cell membrane ofunpermeabilized cells (FIG. 4A, 4B). Interestingly, SAS1R wasconcentrated asymmetrically in unpermeabilized transfected cells whereit localized in regions where blebs and lamellipodia were noted by phasecontrast microscopy (arrows). Unpermeable CHO-K1 cells did not stainwith anti-V5 antibody to the C-terminal tag (data not shown), whereaspermeable CHO-K1 did stain (FIG. 4C). The results suggested that theC-terminus of SAS1R is cytoplasmic and the N-terminus is extracellularin orientation. Together, these observations support the conclusion thatSAS1R is a protein that translocates to the plasmalemma. The controlcells transfected with blank vector showed no staining (FIGS. 13A to13C).

Protein-protein interactions between SAS1R and SLLP1: SPR analysis: Asnoted earlier, SAS1R was first identified by SPR as a putative partnerprotein for the sperm acrosomal ligand SLLP1 (FIGS. 9A and 9B).

Far-western analysis: Affinity between SAS1R and SLLP1 was studied byfar-western analysis with purified rSAS1R and rSLLP1. Purified rSAS1Rwas resolved by SDS-PAGE, stained with Coomassie (FIG. 5 , FW1) and alsotransferred to nitrocellulose membranes. A blot probed with C-terminalanti-His tag monoclonal antibody, identified the specific rSAS1R bands(FIG. 5 , FW2), including the full length and truncated proteins.Additional blots were either overlaid or not with rSLLP1, washed andprobed with monoclonal antibody to SLLP1 (FIG. 5 , FW, 3, 4). The FWrevealed that SLLP1 interacted strongly with the full length SAS1Rdoublet (˜51, ˜50 kD) but very weakly to the C-terminal fragment (˜25kD) [of equal intensity], indicating that the N-terminus of SAS1R isimportant to the conformation of the SLLP1 binding domain than theC-terminus alone.

Yeast two hybrid (Y2H) analysis: The Y2H system under stringentselection conditions was used to study the affinity between SLLP1 andthree SAS1R constructs: full-length 414 residues (V2; SEQ ID NO:6),N-terminal 121 residues, and C-terminal 210 residues. Successfulco-transfection by both plasmid constructs was confirmed by survival onthe low stringency plate (FIG. 14 ). Protein-protein interactions wereconfirmed by survival and formation of blue colonies on high stringencyplates. Yeast cells co-transfected with SLLP1 and N-terminal SAS1Rshowed the fastest rescue with strongest blue color by 3 and 5 days(FIG. 5D, 14D). Co-transfection of C-terminal SAS1R and SLLP1 alsorescued the cells within three days (FIG. 14E, HS); however,co-transfection of full length SAS1R with SLLP1 did not rescue the yeastcells in 3 days but only weakly in 5 days (FIG. 5C, 14 ). The relativelack of full-length SAS1R interaction with SLLP1 is likely due to thepresence of the putative transmembrane domain in SAS1R as membraneproteins are known to be incapable of nuclear transport and properfolding leading to weaker or no interaction in the yeast system (19,20). The results in the Y2H system confirm the strong molecularinteraction between N-terminal SAS1R and SLLP1.

Co-Immunoprecipitation (co-IP) studies: To further study interactionsbetween SAS1R and SLLP1, co-IP was performed with ³⁵S-methionine labeledrecombinant constructs. The in vitro translated N-, C-, full lengthSAS1R and p53 contained myc-tags while SLLP1 and T-antigen had HA-tagsand each produced a major band at expected masses (FIG. 15 ). SLLP1 (˜16kD) was pulled down by co-IP with anti-myc tag antibody to SAS1R fulllength, N- and C-terminal fragments (FIG. 6A). In the reverse co-IP,SAS1R full length (˜50 kD), N- (˜21 kD) and C-terminal (˜26 kD)fragments were pulled down by anti-HA antibody to SLLP1 (FIG. 6B). TheIPs of SLLP1 and SAS1R constructs were performed with HA-tag and myc-tagantibodies, respectively. As a positive control, the T-antigen waspulled down by co-IP with p53 tagged myc antibody. Together, the resultsindicate that SLLP1 has interacting domains with SAS1R.

Co-localization of SAS1R and SLLP1: To determine if SAS1R and SLLP1co-localize on gametes, M2 oocytes or capacitated sperm were incubatedwith each purified recombinant proteins, washed, and probed with bothantibodies. SAS1R signals were present strongly in the microvillarregion of the oolemma and weakly in PVS of the oocyte, and co-localizedwith the major SLLP1 signal, which also diffusely stained the PVS (FIG.7A). The diffuse staining of SLLP1 and SAS1R in the PVS is consistentwith the presence of oolemmal microvilli and CD9 in PVS of M2 oocytes(21). Conversely, SLLP1 localized in the anterior acrosomal region ofsperm, and the SAS1R binding sites were observed to preciselyco-localize with SLLP1 (FIG. 7B). In sum, native SAS1R co-localizes withrSLLP1 binding sites on the oocyte membrane and native SLLP1co-localizes with rSAS1R binding sites on the sperm acrosomal membraneindicating shared domains of specific interaction.

rSAS1R inhibits in vitrofertilization (IVF): To determine the role ofmouse SAS1R during IVF, capacitated spermatozoa were pre-incubated withrSAS1R prior to insemination (FIG. 8 ). A statistically significantreduction in fertilization was observed in samples treated with 25 μg/ml(89% reduction, P<0.03) or more of SAS1R. While a reduction was noted at10 μg/ml, this was not statistically significant. When sperm wereincubated with no protein, fertilization was not reduced. Theseobservations are in agreement with the previous inhibition of mouse IVFby rSLLP1 (2). Together, the results suggested a role of SAS1R infertilization.

Verification of Results Using Native SAS1R and Native SLLP1

Further experiments were done showing that native SLLP1 and native SAS1Rinteract with one another. For example, it was shown that native mSLLP1binds to mSAS1R microvillar domain in a co-localization study in zonaintact mouse MII oocytes. In that study, native mouse cauda spermextract in PBS was incubated with zona intact mouse M2 egg and probedwith guinea pig anti-SAS1R or rat anti-SLLP1 immune or pre-immune serafollowed by probing with fluorescent conjugated secondary antibodies(data not shown).

In another study using native proteins, it was demonstrated usingco-localization techniques that native acrosomal mouse SLLP1 binds toSAS1R in zona intact oocytes (data not shown).

In yet another study, the interaction of native mSAS1R and native mSLLP1was further verified using co-immunoprecipitation techniques and a ratantibody directed against mSLLP1, using a sperm-egg extract, followed bywestern analysis using a guinea pig antibody directed against mSAS1R. Itwas found that following mixing sperm and egg extracts, that theanti-SLLP1 antibody was able to precipitate a complex of SAS1R andSLLP1, as demonstrated in the western blot (data not shown).

These experiments provide further support for those described above.

DISCUSSION

SAS1R is an oolemmal SLLP1 receptor: Five lines of biochemical evidencesupport the hypothesis that SAS1R is an oolemmal receptor for theintra-acrosomal ligand SLLP1. First, native SAS1R extracted from oocytesspecifically bound to and was eluted from rSLLP1 bait from SPR chip(FIGS. 9A and 9B). Second, rSLLP1 bound to rSAS1R in far westernanalyses (FIG. 5 ). Third, molecular affinity between SAS1R and SLLP1was demonstrated in the Y2H assay system (FIG. 5 ), a eukaryotic model.Fourth, SAS1R-SLLP1 interactions were shown by co-IP of SAS1R withantibody against SLLP1, and the converse, using eukaryotic in vitrotranslation system (FIGS. 6A and 6B). Fifth, co localization of rSLLP1binding and SAS1R expression profile on oocyte membrane (FIG. 7A) wasconsistent with the binding of rSLLP1 to native SAS1R (FIGS. 9A and 9B),while the converse study demonstrated the co-localization of rSAS1R andSLLP1 on the acrosomal membrane of capacitated sperm (FIG. 7B). Theoolemmal localization of SAS1R in mature oocytes is also supported bysurface localization of SAS1R in unpermeabilized CHO-K1 cells (FIGS. 4Ato 4C). Furthermore, the rSAS1R/rSLLP1 binding to opposite gametes isalso strengthened by their ability to inhibit fertilization of cumulusintact oocytes (FIG. 8 , (2)). Noteworthy, both SLLP1 and SAS1R aregamete specific molecules and both are exposed prior to sperm-eggfusion. Both Far-western and Y2H studies suggest that the SAS1RN-terminus binds SLLP1 more strongly than the C-terminus. Together theselines of evidence support the hypothesis that SAS1R is an oolemmalreceptor that binds the sperm ligand SLLP1.

SAS1R is an oocyte specific oolemmal protein: EST databases showexpression of SAS1R exclusively in the ovary, oocyte, and zygote in micesuggesting a tight spatial and temporal regulation of SAS1R geneexpression. In mouse ovary, immunolocalizations showed the SAS1R proteinis confined to ooplasm of primary, secondary and Graafian follicles.Thus, available evidence both at protein and mRNA levels supports theconclusion that SAS1R is specifically expressed only in the oocyte andearly embryo, suggesting it is a maternal gene expressed duringoogenesis.

Dynamic re-localization of SAS1R to the microvillar domain: SAS1R waslocalized throughout the ooplasm of germinal vesicle stage oocytes andwas particularly concentrated symmetrically in a corona at the oocyteperiphery. SAS1R subsequently localized to the microvillar domain of theoolemma in M2 arrested ovulated oocytes, being detected at the cellsurface of live oocytes. These observations indicate that a dynamicre-organization of the SAS1R domain occurs as the oocyte maturesfollowing meiosis I and the two distinct regions of the oocyte plasmamembrane form; i.e., the amicrovillar region overlying the eccentricmeiotic spindle, and the microvillar region (6). The development of thispolarity with respect to SAS1R is particularly noteworthy, as sperm bindto and fuse with the oolemma in the microvillar region.

Antibodies generated to SAS1R in both mice and guinea pigs localized theprotein on the microvillar domain in both zona-intact and zona-freelive, ovulated oocytes, indicating that the molecule is exposed at thecell surface (see FIGS. 16 and 17 ). Further, CHO-K1 cells transfectedwith full length SAS1R (FIGS. 4A to 4C) showed localization to plasmamembrane protrusions rather than the planar portions of the cellsurface. This observation directly correlates with published reportsregarding targeting of membrane proteins to sites of membrane insertionin microvilli of epithelial cells and to plasma membrane protrusions ofnon-epithelial cells (22). The observation that unpermeabilized CHO-K1cells did not stain with antibody to C-terminal V5 tag but by anti-SAS1Rantibody, whereas permeable cells did stain by both, suggests that theC-terminus of SAS1R is not accessible externally at the cell surface.This result is consistent with the notion that the N-terminus of SAS1Ris exposed on the cell surface and mediates interaction with the SLLP1ligand. Furthermore, the stronger interactive nature of N-terminus ofSAS1R with SLLP1 is also evident from both Far-Western and Y2H analyses(FIG. 5 ).

Role of SAS1R in sperm oolemma binding: The specific microvillarlocalization of SAS1R in mature oocytes and co-localization of rSLLP1 tothis region (FIGS. 7A and 7B), which is known to be involved insperm-egg binding and fusion (23), provides correlative physiologicalsupport for SAS1R-SLLP1 binding interactions in vivo. An earlier reportshowed that rSLLP1 reduced fertility and sperm-egg binding at the levelof oolemma in a dose dependent manner in mouse IVF studies and retentionof SLLP1 in 90% of acrosome reacted sperm (2; see also U.S. patentapplication Ser. No. 11/915,225, filed Nov. 24, 2008). The present studyalso revealed a dose dependent inhibition of fertilization when spermwere incubated with rSAS1R (FIG. 8 ). These data, together with themolecular interactions between SAS1R and SLLP1 evident from surfaceplasmon resonance, Far-western, Y2H and co-IP studies, suggest SAS1Rfunctions as a receptor for intra-acrosomal SLLP1 at the oolemma.

SAS1R is an active protease: Bioinformatic analysis predicted that SAS1Rbelongs to a metalloprotease family due to its characteristic zincbinding active site signature. The observation of in vitro proteolyticactivity of rSAS1R confirmed this prediction experimentally (FIGS. 1Aand 1B, 11 ). Several other metalloproteases have been reported in theovary and their role in ovulation and formation and regression of corpusluteum has been noted (24). These metalloproteases were localized eitherin theca interna or externa, interstitial glands, germinal epithelium,in granulosa or theca-interstitial cells and in oocyte cytoplasm (25).By contrast, SAS1R is the first metalloprotease reported to beselectively expressed in oocytes and to be localized on the oolemmalsurface. These properties along with the known drugability ofmetalloproteases suggest this molecule as a candidate contraceptivetarget. To our knowledge, there are no previous reports of a mammalianoolemmal metalloprotease. Metalloproteases are known to be involved incellular fusion events. In yeast, cell-cell fusion requires the zincmetalloprotease gene, AXL1 (26). In sea urchin, the zinc chelator(1,10-phenanthroline) showed no effect on binding of acrosome reactedsperm to the oolemma but virtually blocked (95%) subsequent membranefusion (27). In mouse, inhibitors of the aspartic, cysteine, and serineprotease classes had no effect on sperm egg binding or fusion. However,1,10-phenanthroline, a metalloprotease inhibitor, inhibited mousesperm-egg fusion without reducing sperm-egg binding, suggesting acritical role of membrane metalloproteases in gamete fusion (28).

SAS1R conservation in mammals and role in fertilization: SAS1R has beenidentified and characterized in the mouse model as an oolemmal receptorfor the sperm acrosomal ligand SLLP1. Orthologous and homologous geneswith significant homology were identified in other mammals and in lowerorganisms, respectively. Those in Drosophila and C. elegans showed about36% identity while zebrafish showed 42% identity to mouse SAS1R. Allthese species conserve the signal peptide and the zinc bindingmetalloprotease domain (FIGS. 10A and 10B). The putative transmembranedomain appears to be conserved in mammals but notably absent in bird,fish, and invertebrates, except Drosophila, where it is expressed in thegonad.

In lower organisms, homologous proteins were characterized as hatchingenzymes (29). In a previous report an astacin metalloproteinase detectedat the RNA level in human and mouse ovaries was tentatively calledovastacin, and, based on evolutionary conservation of function, asimilar role in zonal hatching was predicted in mammals (30). Theexpression of SAS1R begins to decrease after fertilization and isvirtually undetectable in blastocysts prior to hatching (FIG. 3 ) whileSAS1R RNA was not detected beyond 1.5 days postcoitum in preimplantationembryos (30). Taken together these results point to a role of thisprotein in mammalian fertilization, particularly sperm-oolemmainteractions, rather than in zonal hatching in mammals, suggesting thatthe name SAS1R (sperm acrosomal SLLP1 receptor) best conveys itsbiological interactions.

Fertilization in mammals culminates with fusion of sperm with the oocytemembrane. After acrosome reaction at the zonal surface, the inneracrosomal membrane of the sperm becomes exposed and serves as thelimiting membrane on the anterior sperm surface. The intra-acrosomalprotein SLLP1 is exposed and retained on the inner acrosomal membrane ofacrosome reacted sperm (2). Thus, acrosome reacted sperm that penetratethe zona pellucida will display SLLP1 on their anterior heads andequatorial segments and interact with microvillar SAS1R. The presentstudy advances our understanding of molecular interactions betweenacrosome-reacted sperm and the mature oocyte at the level of theoolemma.

Example 2-SAS1R as a Target for Contraception

Effect of SAS1R antibody on mouse in-vitro fertilization. Antibodies andsera directed SAS1R were prepared. Cumulus intact mouse oocytes wereincubated with either preimmune or immune sera at 1/10 & 1/20 (N=4) or1/80 (N=2) dilutions for 45 min followed by insemination withcapacitated sperm. The number of two cell embryos was scored asfertilized eggs. The statistical significance between the preimmune andimmune sera was calculated by t test assuming equal variances and arepresented in FIG. 16 . It can be seen that an inhibitor of SAS1R, suchas an antibody directed against SAS1R, is an effective inhibitor offertilization.

SAS1R is an Effective Immunogen. Next, it was shown that the SAS1Rprotein is an effective immunogen in females. As an oocyte specific,sperm oolemmal receptor, SAS1R was hypothesized herein to be a candidatecontraceptive vaccinogen and immunogen. Immunogenicity of recombinantmouse SAS1R was tested in female mice which showed serum titers by ELISAagainst the recombinant target up to a 1/10,000 dilution after the 3rd,4th & 5th injections (see FIG. 17 , left panel). The immunoreactivity ofthe sera were also studied by immuno-localization in live mouse eggs(see FIG. 17 , right panel). The iso-antibodies from female mice stainedthe microvillar domain of mouse eggs exactly as noted earlier withallo-antibodies raised in guinea pigs (see right panel). This findingconfirmed that recombinant mouse SAS1R retained sufficient refoldedepitopes to evoke iso-antibodies that cross-reacted with native SAS1R onthe microvillar domain.

Example 3-SAS1R Localization and Temporal Expression Defines theOpportunity for Reversible Contraception

Mouse ovaries were studied from birth until day 56. Sections from day 0(day of birth) neonatal mouse ovaries were obtained and stained withpre-immune or guinea pig anti-SAS1R antibodies at identicalconcentrations [diluted 1:500] (FIG. 18 ). The ovary on this day iscomprised principally of naked oocytes or a few forming primaryfollicles. No immunoreactivity was observed on any ovarian cell typewith either sera on day 0, indicating SAS1R protein was not expressed atthis stage in the ovary.

Sections from day 1.5 mouse ovary were stained with pre-immune atidentical concentrations [1:500] (FIG. 19 ). The ovary on day 1.5 iscomprised principally of primordial follicles and forming primaryfollicles in which oocytes are surrounded by a single flat (squamous)layer of granulose cells. No immunoreactivity was observed on anyovarian cell type with either sera, indicating SAS1R was not expressedat this stage in the ovary.

Sections from day 4 mouse ovary were stained with pre-immune or guineapig anti-SAS1R antibodies at identical concentrations [1:500] (FIG. 20). The cortex of the ovary on day 4 is comprised of primordial andprimary follicles while the ovarian medulla on this day is comprisedprincipally of forming primary follicles in which oocytes are surroundedby a single flat (squamous) layer of granulosa cells as well as moremature secondary follicles containing oocytes surrounded by two layersof cuboidal granulosa cells. Immunoreactivity, indicating the presenceof SAS1R, was observed only in oocytes. Further, and importantly, theonly oocytes that stained had reached the secondary follicle stage.Pre-immune sera did not stain any cell type indicating that the immunestaining was specific for SAS1R. Conclusion: SAS1R is located only inoocytes and only in oocytes that have reached secondary follicle stage.

Sections from day 7 mouse ovary were stained with pre-immune or guineapig anti-SAS1R antibodies at identical concentrations [1:500] (FIG. 21). The ovarian medulla on this day is comprised principally of formingprimary follicles in which oocytes are surrounded by a single flat(squamous) layer of granulosa cells as well as more mature secondaryfollicles containing oocytes surrounded by two layers of cuboidalgranulosa cells Immunoreactivity, indicating the presence of SAS1R, wasobserved only in oocytes. The only oocytes that stained had reached thesecondary follicle stage. Pre-immune sera did not stain any cell typeindicating that the immune staining was specific. Conclusion: SAS1R islocated only in oocytes and only in oocytes that have developed tosecondary follicle stages.

Day 14 mouse ovary sections (14 days after birth) were stained withpre-immune or guinea pig anti-SAS1R antibodies at identicalconcentrations [1:500] (FIG. 22 ). The ovarian medulla on this day iscomprised principally of forming primary follicles in which oocytes aresurrounded by a single flat (squamous) layer of granulosa cells as wellas more mature secondary follicles containing oocytes surrounded by twolayers of cuboidal granulosa cells. A few follicles show signs of beingpre-antral. Immunoreactivity, indicating the presence of SAS1R, wasobserved only in oocytes. The oocytes that stained had all reached thesecondary follicle stage or progressed to the pre-antral stage.Pre-immune sera did not stain any cell type indicating that the immunestaining was specific. The data demonstrate that SAS1R is located onlyin oocytes and only in oocytes that have developed to secondary follicleor pre-antral stages.

Day 28 mouse ovary sections were stained with pre-immune or guinea piganti-SAS1R antibodies at identical concentrations [1:500] (FIG. 23 ).The ovarian cortex on this day is comprised of forming primary folliclesin which oocytes are surrounded by a single flat (squamous) layer ofgranulosa cells, more mature secondary follicles containing oocytessurrounded by two layers of cuboidal granulosa cells, and antralfollicles with many layers of granulosa cells and fluid filled antralspaces. Immunoreactivity, indicating the presence of SAS1R, was observedonly in oocytes. Oocytes that stained were those that had reached thesecondary follicle stage of maturation as well as antral [Graafianfollicle] stages. Pre-immune sera did not stain any cell type indicatingthat the immune staining was specific. Conclusion: In the ovaries ofmice at puberty, SAS1R is located only in oocytes and only in oocytesthat have developed to secondary follicle stage as well as subsequentstages.

Day 56 adult mouse ovary sections were stained with pre-immune or guineapig anti-SAS1R antibodies at identical concentrations [1:500] (FIG. 24). The ovarian cortex on this day is comprised of primordial follicles,primary follicles in which oocytes are surrounded by a single flat(squamous) layer of granulosa cells, more mature secondary folliclescontaining oocytes surrounded by two layers of cuboidal granulosa cells,and many multi-lamellar follicles including antral follicles with manylayers of granulosa cells and fluid filled antral spaces.Immunoreactivity, indicating the presence of SAS1R, was observed only inoocytes. Oocytes that stained were those in secondary follicles as wellas all intermediate sized oocytes as well as antral [Graafian follicle]stages. Pre-immune sera did not stain any cell type indicating that theimmune staining was specific. Particularly noteworthy is the absence ofstaining in corpora lutea. Conclusion: In the ovaries of adult mice,SAS1R is located only in oocytes and only in oocytes that have developedto secondary follicle stage. The SAS1R protein then persists in theoocyte cytoplasm in follicles of all sizes including ovulatingfollicles.

Summary

Evidence that SAS1R is a Receptor for SLLP1

Native SAS1R showed binding to rSLLP1 by surface plasmon resonancetechnique.

Bound rec SAS1R captured rec SLLP1 in membrane overlay assay (FarWestern analysis).

SAS1R and SLLP1 revealed molecular binding properties by yeast twohybrid analysis.

Immunoprecipitation of rSAS1R recovered rSLLP1 and immunoprecipitatedrec SLLP1 recovered rec SAS1R from rabbit reticulocyte extract.

rSLLP1 binds to oocyte microvillar domain and co-localizes with nativeSAS1R.

rSAS1R binds to acrosome of sperm and co-localizes with native SLLP1.

Native SLLP1 from sperm acrosomal matrix localizes with native SAS1R.

Native SAS1R and native SLLP1 are co-precipitated from mixtures ofnon-ionic detergent extracts of oocytes and sperm.

Contraceptive Target and Use Implications of Data on SAS1R Ontogeny

SAS1R protein first arises in bilaminar secondary follicles duringpostnatal oogenesis, in pubertal oogenesis, as well as adult oogenesis.The pattern is uniform irrespective of the age of the animal.

In adult mouse ovaries, SAS1R staining is restricted to oocytes withinsecondary follicles and all subsequent stages.

Primordial oocytes and primary oocytes do not stain for SAS1R at anydevelopmental stage. No other cell type but oocytes stain for SAS1R, noris SAS1R found in any other ovarian structure.

SAS1R is localized on live human eggs retrieved for in vitrofertilization.

Administration of exogenous SAS1R elicits an immune response againstSAS1R, which localizes to the egg.

Without wishing to be bound by any particular theory, the data disclosedherein suggest that, regarding contraception, oogonia stem cells,including naked oocytes and oocytes within primordial and primaryfollicles, will not be affected by a drug to SAS1R; the ovarian reserveof stem cells will remain intact.

A contraceptive drug against SAS1R will be reversible.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

BIBLIOGRAPHY

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Example 4

SLLP1 and its Role in Binding to Eggs

Materials and Methods

Cloning and Expression of Mouse SLLP1 (mSLLP1)

Human SLLP1 and SLLP2 nucleic acids and proteins were previouslyidentified and sequenced by the inventors (see U.S. patent applicationSer. No. 10/181,611, filed Jul. 18, 2002, the entirety of which isincorporated herein by reference). These proteins were furthercharacterized, and other members of the family were identified by thepresent inventors (see U.S. patent application Ser. No. 10/542,038 filedJul. 13, 2005, the entirety of which is incorporated herein byreference; see also Mandal et al., 2003, Biology of Reproduction,68:1525-1537, the entirety of which is incorporated herein byreference). Both SLLP proteins are sperm specific in their expression.

Using a Blast search tool (Altschul, 1990), a mouse orthologue of thehuman SLLP1 was sought in the NCBI GenBank database and a candidate geneidentified. Single gene-specific forward and reverse primers with NcoIand XhoI restriction sites respectively were designed to amplify thepredicted processed form (128 amino acids, from 94 to 221) of the mouseSLLP1. Primers were obtained from Invitrogen (Carlsbad, Calif.). ThecDNA was amplified by PCR from a mouse testis cDNA library (Clontech,Palo Alto, Calif.). The cycling parameters employed were 94° C., 2 min;94° C., 30 sec; 51° C., 1 min; and 68° C., 1.5 min, for 40 cycles. PCRreaction products were separated on agarose gels, and a band of ˜400 bpwas isolated, reamplified, and subcloned in pCR2.1 TOPO vector(Invitrogen). Multiple cDNA clones were sequenced in both directionsusing vector-derived primers on a Perkin-Elmer Applied Biosystems DNAsequencer (Biomolecular Research Facility, Univ. of Virginia HealthSystem, VA). The cloned cDNA sequence for mouse SLLP1 was submitted tothe GenBank (accession number, AY601763). The insert was thenrestriction digested, gel purified, ligated into the predigested pET28b+vector and used to transform competent BL21DE3 cells (Novagen, Madison,Wis.). The final construct added two amino acids at the N-terminus andeight residues at the C-terminus including a six histidine tag. A 7.0 mlculture from a single colony was grown to optical density of ˜0.8 at 600_(nm) at 37° C. in Luria broth (LB) in the presence of 50 μg/ml ofkanamycin. Isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma, St. Louis,Mo.) was then added to a final concentration of 1 mM to induceexpression. Following 3 h of induction, the bacteria were collected bycentrifugation. The recombinant protein was isolated from the insolublefraction of E. coli, dissolved in 8 M urea in binding buffer (20 mMtris-HCl, pH 7.9, 5 mM imidazole and 0.5 M NaCl) and purified on a Hisbinding Ni²⁺ chelation affinity resin column by a modification of themanufacture's procedures (Novagen). The eluates were then dialyzedovernight against three changes of PBS. The dialyzed protein was storedat −20° C. until used. Protein concentrations were determined byCoomassie Plus-200 (Pierce, Rockford, Ill.) using bovine serum albumin(BSA) as a standard.

Polyclonal Antibody Production and Western Blot Analysis

Five adult virgin female guinea pigs were used for antibody productionagainst the purified recmSLLP1. Preimmune serum was collected by heartpuncture, and subsequently, each animal was injected with 200 μg of thepurified recmSLLP1 in complete Freund's adjuvant and boosted twice atintervals of 14 days with the same amount of protein in incompleteFreund's adjuvant. For all immunizations, half of the antigen emulsionwas injected intramuscularly in the legs and half subcutaneously in twosites on the back. All animals were exsanguinated by heart puncture 9days after the final immunization. Blood was collected in serumseparation tubes (Becton Dickinson, Franklin Lakes, N.J.). Aftercentrifugation at 1750 g for 10 min, the serum was removed, aliquoted,and frozen until needed.

Specificity of the antisera was tested against recmSLLP1 and mouse spermextracts following 1D SDS-PAGE western blotting. RecmSLLP1 (0.1 μg/lane)or cauda epididymal mouse spermatozoa (10 μg/lane) were solubilized inLaemmli buffer (2×) and proteins were resolved on a 15% SDS-PAGE gel andseparated at 20 mA. Proteins were then blotted to nitrocellulose andstained by Ponceau. All blots were blocked with 5% nonfat dry milk inPBS with 0.05% Tween 20 (PBS-T) for 30 min at room temperature. Forimmunoblotting of purified recmSLLP1, 1:15,000 or 1:30,000 dilution ofthe anti-recmSLLP1 guinea pig sera was tested, whereas for mouse spermproteins, 1:5,000 or 1:10,000 dilutions of the sera was used. The blotswere then washed three times for 10 min in PBS-T, and incubated with1:5000 dilution of peroxidase conjugated goat anti-guinea pig IgGsecondary antibody for 1 h and washed two times for 10 min in PBS-T. Theblots were then developed either in TMB peroxidase substrate(3,3′,5,5′-tetramethylbenzidine, KPL, Gaithersburg, Md.) or with ECLreagent (Amersham Corp., Buckinghamshire, UK).

Culture Media and Reagents for In Vitro Fertilization Assays

The medium used for in vitro fertilization assays was Fraser'smodification of Whittingham's medium (Fraser and Drury, 1975)supplemented with 3% BSA and prepared with culture grade H₂O withanalytical-grade reagents. TYH (Toyoda, 1971) medium was used forsperm-oolemma binding assays. Pregnant mare's serum gonadotrophin(PMSG), human chorionic gonadotrophin (hCG), BSA, culture grade H₂O,hyaluronidase, chymotrypsin, Hoechst dye 33342 and other reagents wereobtained from Sigma.

Gamete Preparation for In Vitro Assays

Hybrid F1 mice (C57BL/6J×CBA) were used in all experiments. Suspensionsof epididymal spermatozoa from sexually mature male mice were preparedfor insemination of isolated oocytes. Oocytes were obtained from28-day-old females superovulated with 10 IU PMSG and 10 IU hCG, injectedintraperitoneally 48 h apart. Females were killed 16 h after hCGinjection and both oviducts were immediately removed and placed inmineral oil.

In Vitro Fertilization with Cumulus-Oocyte Complexes

In vitro fertilization with cumulus intact oocytes was conducted withsperm dispersed from cauda epididymides placed for 5 min in 200 μl dropsof fertilization medium under paraffin oil. The sperm suspension wasdiluted to a concentration of 10⁶ sperm/ml in a volume of 200 μl andthen incubated for 120 min in a humidified tissue culture incubator (37°C., 5% CO₂ in air) to allow capacitation. In the experiments whereanti-recmSLLP1 serum was tested, spermatozoa were incubated with varyingconcentrations of decomplemented (56° C., 30 min) immune or preimmuneserum for the last 45 min of capacitation. In the experiments whererecmSLLP1 was evaluated, spermatozoa were incubated under standardcapacitating conditions.

Cumulus masses were placed in 135 μl drops of fertilization medium (onemass per drop) under paraffin oil and were incubated for 45 min withimmune or preimmune serum or in presence or absence of recmSLLP1 priorto insemination. Fifteen μl of the sperm suspension (finalconcentration: 10⁵ sperm/ml) was then added to each cumulus mass drop.Thus, sera or recombinant protein was present in the incubation dropletduring gamete interaction. Six hours following insemination oocytes wererelocated in 100 μl drops of fertilization medium under mineral oil.Following overnight incubation, eggs were stained in Hoechst dye (10μg/ml) for 10 min and washed 3 times in fertilization medium. The eggswere then placed in a 5 μl drop of fertilization medium between amicroscope slide and an elevated coverslip, and visualized at 160× usinglight and fluorescence microscopy (Zeiss Axioplan). Two cells embryoswere scored as fertilized, while one-celled oocytes were scored asunfertilized.

In Vitro Fertilization with Zona-Free Eggs

For the sperm-oolemma binding assay, two cauda epididymides were placedin 900 μl drops of fertilization medium under paraffin oil for the densemass of spermatozoa to flow freely for ˜15 min and then diluted at1×10⁶/ml for 3 h of capacitation. Cumulus oocyte complexes were placedin 200 μl drops of TYH medium under paraffin oil. Cumulus cells wereremoved by treating the oocytes for 3 min with 1 mg/ml hyaluronidase inTYH medium and then washed 8 times in 50 μl drops. Zona pellucidae wereloosened by treating the oocytes with 10 μg/ml chymotrypsin in TYH mediafor 1 min and loosened zonae were removed by mechanical agitation usinga pulled Pasteur pipette. The oocytes were then washed 10 times andallowed to recover from chymotrypsin treatment by incubating in TYHmedia for 3 h, following which, they were stained with 10 μg/ml Hoechstdye for 10 min, and then gently washed.

To test the effects of anti-recmSLLP1 antibodies, spermatozoa wereincubated with varying concentrations of decomplemented immune orpreimmune sera for the last 30 min of capacitation. Untreated oocyteswere then added to the incubation drops containing the treated spermwith the final concentration being 2.5×10⁴ sperm/ml.

To compare the effects of recmSLLP1 with human and chicken c lysozymes,spermatozoa were first incubated under standard capacitating conditions.Oocytes were pre-incubated for 45 min before insemination with recmSLLP1(0.1 to 200 μg/ml) or with hSLLP1 (25 μg/ml) or with chicken or humanlysozyme (50 μg/ml, 100 μg/ml). Untreated capacitated sperm were thenadded to the incubation drops containing the treated eggs with a finalsperm concentration of 2.5×10⁴/ml. Thus, in all the experimentsperformed the sera, the recombinant protein, or the lysozyme was presentin the incubation droplet during gamete interaction. After 30 min ofgamete co-incubation, oocytes were gently washed 5 times in TYH mediumand placed between a microscope slide and an elevated coverslip andvisualized at 160×. Binding to the oocyte was scored by counting thenumber of bound spermatozoa per oocyte using phase contrast. Fusion withthe egg was scored by counting the number of decondensed sperm headswithin each oocyte using fluorescence microscopy.

Indirect Immunofluorescence Studies of Mouse Spermatozoa and OocytesLocalization of mSLLP1 on Fixed Spermatozoa

Cauda epididymal mouse spermatozoa were placed in 0.9 ml drop ofphosphate-buffered saline without calcium (PBS; pH 7.4) (twoepididymides per drop) and incubated at 37° C. in an atmosphere of 5%CO₂ for 5 min. To induce the acrosome reaction, spermatozoa wereincubated in TYH media for 90 min to undergo capacitation (Visconti etal, 1995) and 5 μM calcium ionophore A23187 was added for another 15 minfor the acrosome reaction to occur. Each drop was then collected,centrifuged for 10 min at 500 g and resuspended in PBS; this washingprocedure was repeated three times. Smears of the final suspension ofmouse sperm in PBS were air-dried on microscope slides at roomtemperature and fixed in 2% w/v paraformaldehyde in PBS for 10 min.After 6 washes in PBS, spermatozoa were incubated for 30 min at 37° C.with normal goat serum (NGS) (5% v/v in PBS) and then incubated for 1 hwith anti-recmSLLP1 sera (1:25). The slides were washed 3 times in PBSand spermatozoa were incubated for 1 hour at 37° C. with Texasred-conjugated polyclonal antibody from donkey (1:200, JacksonLaboratories). Slides were then washed, incubated for 30 min at roomtemperature with Peanut agglutinin lectin (PNA) (1:50) (MolecularProbes) conjugated with FITC, washed, mounted in Slowfade® (MolecularProbes, Eugene, Oreg.), and visualized under a Zeiss Standard 18ultraviolet microscope. Images were captured by using MrGrab (Carl ZeissVision GmbH, Germany).

Egg labeling: Metaphase II eggs were obtained as previously described(Coonrod et al, 1999) and incubated with 5% NGS/media for 30 min.Oocytes were washed five times in TYH medium and incubated with 100μg/ml recmSLLP1 or 100 μg/ml lysozymes for 45 min at 37° C. and 5% CO₂.Oocytes were washed five times and incubated with guinea piganti-recmSLLP1 polyclonal antibody (1:50), sheep anti-human lysozyme(1:25) or rabbit anti-chicken lysozyme (1:400) in 5% NGS/media for 1 hat 37° C. and 5% CO₂. Oocytes were washed five times and incubated withdonkey anti-guinea pig/Texas Red antibody (1:200) or goat anti-guineapig/FITC antisera 1:200), donkey anti-sheep and goat anti-rabbitFITC-labeled secondary antibody (1:200) (Jackson ImmunoResearch),respectively in 5% NGS/media for 1 h at room temperature at 37° C. and5% CO₂. Oocytes were washed and mounted in media onto glass slides andvisualized under a Zeiss Standard 18 ultraviolet microscope. Images werecaptured by using MrGrab 1.0 (Carl Zeiss Vision GmbH, Germany).

Scanning Confocal Microscopy

Metaphase II eggs employed for immunofluorescence studies (above) wereutilized for scanning confocal microscopy. The stained eggs were washedthree times in PBS containing 1% BSA (PBS/BSA) and then fixed in 4%paraformaldehyde in PBS-polyvinylalcohol (PVA) for 20 min at roomtemperature. Following fixation, eggs were washed 5 times in PBS/BSA andthen permeabilized with 0.5% Triton X-100 in PBS for 20 min at roomtemperature. Eggs were then washed five times in PBS/BSA and placed in0.4 mg/ml RNase in PBS/BSA for 30 min and then stained with 20 nM Sytox(Molecular Probes) for 10 min. Eggs were then extensively washed, placedin slow fade (Molecular Probes) equilibration media for approximately 1min and then mounted on slides in slow fade mounting media. Images wereobtained on a Zeiss 410 Axiovert 100 microsystems LSM confocalmicroscope. For each panel, attenuation, contrast, brightness andpinhole aperture remained constant. For each panel, four seconds scanswere averaged four times per line using a 63× oil lens equipped with azoom factor of two.

Sperm Labeling During Binding to Metaphase II Eggs

Zona-free eggs inseminated with capacitated spermatozoa from the invitro fertilization studies above were fixed with 2% paraformaldehydefor 10 min at room temperature. Gametes were washed in PBS-BSA,incubated with 5% NGS/PBS-BSA for 30 min at 37° C. and then incubatedfor 1 h with anti-recmSLLP1 sera (1:25). The slides were washed 3 timesin PBS and gametes were incubated for 1 h at 37° C. with donkeyanti-guinea pig Texas red-conjugated polyclonal antibody (1:200, JacksonLaboratories). Gametes were then washed five times in PBS/BSA, placed in0.4 mg/ml RNase in PBS/BSA for 30 min, and then stained with 20 nM Sytox(Molecular Probes) for 10 min for nuclear staining. Gametes wereextensively washed, placed in slow fade (Molecular Probes) equilibrationmedia for approximately 1 min and then mounted on slides in slow fademounting media. Images were obtained on a Zeiss 410 Axiovert 100microsystems LSM confocal microscope as described above.

Statistical Analysis

All in vitro assays were repeated at least three or more times.Experimental and control group values were reported as means±standarderror of mean. Groups were compared using the matched-pairs t-testassuming equal variances and differences were reported at P≤0.05 as thelevel of significance (Bowers, D. Medical Statistics from Scratch. JohnWiley & Sons; West Sussex, U K, 2002, pp. 129-132).

Results

Mouse SLLP1 is the True Orthologue of hSLLP1 and Shares SimilarCharacteristics to c Lysozymes

The complete amino acid sequence of mSLLP1 was deduced (predicted mol.wt. 25 kDa, pI—6.2). The N terminus of mSLLP1 contains a predictedtransmembrane domain followed immediately by a potential proteasecleavage site between alanine 93 and lysine 94 linkage. Comparison ofthe full-length hSLLP1 and mSLLP1 sequences using the Accelrys Gap(Seq/Web version 2) algorithm found that mSLLP1 is 64.2% similar and58.8% identical to the hSLLP1. The mSLLP1 processed form, starting afterthe protease cleavage site (128 amino acids), shares 82.8% similarityand 75.8% identity to that of hSLLP1. The deduced mSLLP1 sequencecontains three putative myristoylation sites (G2, G41 and G142),potential phosphorylation sites for casein kinase II (S97) and proteinkinase C (S66, S90, S152, and S153) and a signature sequence for thealpha-lactalbumin/lysozyme C family. The predicted molecular weight(14.6 kDa) and pI (5.2) of mature mSLLP1 are identical to hSLLP1.

In addition, a Blast search of the NCBI GenBank database and a multiplesequence alignment of selected mature c lysozymes revealed that maturemSLLP1 is 46%, 48% and 50% identical to mouse, human and chickenlysozymes, respectively. Forty-one residues in mSLLP1 are identical tothe three conventional lysozymes. Among the 20 invariant residues of clysozymes (Prager, E. M., and Jolles, P., Animal lysozymes c and g: anoverview. In: Jolles, P. (Ed.), Lysozymes: Model Enzymes in Biochemistryand Biology. Birkhauser Verlag, Basel, 1996, pp. 9-31), 16 were found tobe conserved in mSLLP1. Interestingly, the essential catalytic residues(E35 and D52) of chicken lysozyme were replaced with T35 and N52 inmSLLP1 as well as hSLLP1 (Prager, E. M., and Jolles, P., Animallysozymes c and g: an overview. In: Jolles, P. (Ed.), Lysozymes: ModelEnzymes in Biochemistry and Biology. Birkhauser Verlag, Basel, 1996, pp.9-31). Among the six potential substrate-binding residues of clysozymes, five were conserved in both mSLLP1 and hSLLP1 (Kumagai, I.,Sunada, F., Takeda, S., and Miura, K., 1992. Redesign of thesubstrate-binding site of hen egg white lysozyme based on the molecularevolution of C-type lysozymes. J. Biol. Chem. 267, 4608-4612).

The mouse SLLP1 gene, Spaca3, is a six exon gene located on chromosome11 at locus B5 where it is flanked by the gene for amiloride-sensitivecation channel 1, Accn1, and an unknown protein belonging to the myosinfamily. This locus in the mouse is considered syntenic with 17q12 wherethe human SLLP1 gene, SPACA3, is also flanked by ACCN1 and the myosingene MYOID. Furthermore, the intron positions of mature mouse and humanSLLP1 precisely match with that for human and mouse lysozymesinterrupting codons for Trp, Asp/Ala and Trp, suggesting a possibleorigin of these genes from a common ancestor.

Expression of mSLLP1 and Specificity of the Antibody

A cDNA sequence encoding the mature mSLLP1 from residue 94 to 221(beginning after the putative protease cleavage site), and including asix-histidine C terminal tag was expressed in E. coli and a recombinantprotein of about 15 kDa was obtained after Ni⁺⁺-affinity purification.To evaluate the relative purity of the recmSLLP1 preparation, an aliquotof the purified protein was separated by 1-D electrophoresis and the gelwas silver stained and blotted to probe with anti-his antibody. Aprominent band of about 15 kDa and a much fainter putative dimer atabout 30 kDa were noted. These results indicated that the recmSLLP1preparation used for this study was highly purified.

The specificity of the antibody generated in guinea pigs againstrecmSLLP1 was examined by western blotting against both the recombinantimmunogen and mouse sperm proteins. The immune sera recognized the 15kDa recombinant SLLP1 as well as the putative 30 kDa dimer found in therecombinant preparation, while the preimmune serum as well as the serumfrom guinea pigs injected with adjuvant alone showed no immunoreactivitywith recSLLP1. In mouse sperm extracts the immune serum reacted onlywith a about 15 kDa band while the serum from guinea pigs injected withadjuvant alone as well as preimmune sera showed no reactivity. Theseresults indicated that a specific immunoreagent had been generated tothe recmSLLP1 that gave a single band on sperm protein extracts. Findingonly a ˜15 kDa form of mSLLP1 in sperm protein extracts demonstratedthat full length mSLLP1, predicted to run at ˜25 kDa, was not detectablein sperm. Further mSLLP1 dimerization does not occur in sperm although asmall amount does occur during E. coli expression. The observations alsoindicated that E. coli expressed mSLLP1 after affinity purificationcontained sufficient numbers of immunogenic epitopes to generateantibodies cross reactive with the native mSLLP1.

Mouse SLLP1 is Associated with Sperm Acrosome and Retained afterAcrosome Reaction

Indirect immunofluorescence of fixed mouse spermatozoa localized mSLLP1to the anterior acrosome in 70.0% of non-capacitated caudal spermatozoa(Table 1). However, 20.3% of non-capacitated spermatozoa showed anequatorial segment distribution of mSLLP1 and 4.5% possessed no staining(Table 1). Seventy-one percent of acrosome-reacted sperm, determined bythe lack of fluorescence in the acrosome by PNA lectin staining,displayed only equatorial segment reactivity with anti-recmSLLP1 serum.However, 13.9% of acrosome-reacted spermatozoa retained an anterioracrosome staining pattern while 9.3% did not stain (Table 1). Thedisappearance of mSLLP1 staining in the anterior acrosome appeared tocorrespond with the appearance of staining in the equatorial segmentfollowing capacitation and the acrosome reaction.

Proteins from acrosome reacted sperm were analyzed by Western analysis(Table 1 insert) using anti-recmSLLP1 serum. Ionophore treated, acrosomereacted sperm showed a ˜14 kD mSLLP1 band with very subtle decrease inmigration compared to proteins extracted from capacitated sperm. Theslower migration of the ˜14 kD band is possibly due to changes inphosphorylation/dephosphorylation of mSLLP1 during the acrosomereaction, a process known to be regulated by protein phosphorylation(Furuya et al, 1992). The presence of mSLLP1 in ionophore treated aswell as capacitated sperm confirmed that the observed equatorialimmunofluorescent staining is a specific SLLP1 pattern. Importantly,confocal analysis showed retention of mSLLP1 in all capacitated mousesperm tightly bound to mouse eggs, emphasizing the concept that thisprotein may be involved in sperm-oolemma binding.

RecmSLLP1 and Anti-recmSLLP1 Serum Inhibit Fertilization of MouseCumulus Intact Eggs

To determine the role of mSLLP1 during fertilization, both spermatozoaand cumulus intact oocytes were pre-incubated with anti-recmSLLP1 serumor preimmune serum for 45 min prior to insemination. Fertilization wasconducted in the presence of the antibody and six hours later the eggswere relocated in 100 μl drops of fertilization medium and incubatedovernight. In the groups treated with the immune sera at 1:10 or 1:50dilutions, the percentage of two cells embryos was significantly (P0.05) reduced (61% and 17% inhibition respectively; Table 2A) from thevalues observed with respective preimmune serum. However, a significanteffect on cumulus intact eggs was not observed at a 1:100 dilution.

Cumulus intact oocytes were also incubated with two concentrations ofrecmSLLP1, which remained in the culture medium during the fertilizationprocess. Treatment of the cumulus-oocyte complexes with 200 μg/mlrecmSLLP1 significantly reduced the fertilization rate from 45% in thecontrol group to 12% in the recmSLLP1 treated group (73% inhibition,P<0.05), whereas no significant difference was observed on thepercentage of fertilization between the control group and the grouptreated with 50 μg/ml recombinant protein, although a reduction wasnoted (Table 2B). Taken together, these results suggested that mSLLP1plays a role in fertilization.

Mouse SLLP1 has a Role in Sperm-Egg Binding

Inhibition of fertilization by recmSLLP1 protein as well as antibodiesto recmSLLP1 using cumulus-egg complexes prompted a dose-ranging studyof the effect of antibody to SLLP1 and recmSLLP1 on gamete binding andfusion to determine the stage in the fertilization cascade at whichmSLLP1 exerted its effects. Therefore, we tested whether anti-recmSLLP1serum and recmSLLP1 protein would block capacitated mouse sperm-eggbinding, fusion, or both to zona-free mouse eggs. Statisticallysignificant inhibition of binding but not fusion was observed when bothgametes were co-incubated in the presence of 1:10 and 1:50 dilutions ofanti-recmSLLP1 immune sera compared to preimmune sera, whereas the 1:100dilutions were not significantly different.

The most striking effect was observed when zona-free mouse eggs wereincubated with different concentrations of recmSLLP1 (0.1-200 μg/ml) andthen inseminated with untreated capacitated mouse spermatozoa. Theincubation of oocytes with recmSLLP1 produced a concentration-dependentdecrease in the number of spermatozoa bound to or fused to the egg, witha significant effect observed at as low as 0.1 μg/ml and 100% inhibitionof both binding and fusion at 200 μg/ml (12.5 μM). Zona-free mouseoocytes incubated in the absence of recmSLLP1 (buffer only) was used ascontrol. Importantly, no difference was observed in the percentages ofmotile spermatozoa compared to the control suggesting thatanti-recmSLLP1 or recmSLLP1 protein did not affect sperm motility butoolemma binding and subsequent fusion. Taken together, these resultssupport the participation of mSLLP1 in the binding event at the mouseegg surface prior to fertilization.

Mouse SLLP1 has Complementary Binding Sites on Unfertilized andFertilized Oocytes

To study the possible localization of mSLLP1-binding sites on the eggsurface, unfertilized oocytes along with in vitro fertilized oocytes atthe pronuclear stage, were incubated with purified recombinant mSLLP1for 45 min, washed, and then exposed to anti-recmSLLP1. Unfertilizedoocytes exhibited fluorescent labeling within the perivitelline spaceand over much of the oocyte surface. However, an area devoid offluorescence was consistently detected. Hoechst staining revealed thatthis negative area was always associated with the area of the oocyteplasma membrane overlying the metaphase plate. Thus, mSLLP1-bindingsites were restricted to the fusogenic region of the egg, consistentwith a role for mSLLP1 interaction in sperm-egg binding. Interestingly,oocytes, with or without zona pellucida, that had been fertilized invitro and treated with recmSLLP1, exhibited intense, patchyimmunofluorescent domains over the entire egg surface, indicating thatSLLP1 binding sites remain associated with egg surface domains afterfertilization. Controls included oocytes not exposed to recmSLLP1 andthen exposed to anti-recmSLLP1, oocytes incubated with recmSLLP1 andthen exposed to preimmune sera, and oocytes incubated with recePAD (anegg cytoplasmic protein; Wright et al., 2003, ePAD, an oocyte and earlyembryo-abundant peptidylarginine deiminase-like protein that localizesto egg cytoplasmic sheets. Dev. Biol. 256, 73-88) and then incubatedwith the respective specific antibody. None of these three controlsshowed egg surface fluorescence.

Confocal analyses were then employed to refine the localization ofmSLLP1 binding sites in the egg after treatment with recombinant SLLP1.Optical sections of zona intact unfertilized oocytes showed thatmSLLP1-binding sites were localized predominantly to the perivitellinespace. In contrast, fluorescence was virtually undetected in theperivitelline space of fertilized eggs while an intense signal for SLLP1was evident on the oolemma in distinct patches. A weak fluorescentlabeling was also observed on zona pellucidae of both unfertilized andfertilized eggs.

C Lysozymes do not Block Gamete Binding or Fusion to the Mouse Egg

Mouse and human SLLP1 are lysozyme-like proteins that share severalcharacteristics of the c lysozyme family. Mouse SLLP1 is 48% identicalto human and 50% identical to chicken lysozyme, 46% identical toconventional mouse lysozyme, while human SLLP1 is 52% identical to humanlysozyme and 48% identical to chicken lysozyme. To determine whetherconventional c lysozymes have an inhibitory effect similar to SLLP1 onsperm-egg binding and fusion, human and chicken lysozyme were incubatedwith mouse oocytes at concentrations shown previously to exert maximaleffects for SLLP1s. Although 50 μg of mouse SLLP1 and 25 μg of humanSLLP1 maximally inhibited sperm-egg binding and fusion, a similar effectwas not observed even at 50 or 100 μg/ml of the conventional clysozymes. Similarly, mouse oocytes did not show any fluorescence whenincubated with human or chicken c lysozymes and their respectiveantibodies, indicating a lack of oolemmal receptors for these clysozymes.

Table 1: Incidence of SLLP1 staining patterns in populations of acrosomeintact and acrosome reacted mouse spermatozoa. In acrosome intact sperm,defined by positive PNA staining, mSLLP1 localized mainly to theanterior acrosome (70.0%) secondarily to the equatorial segment (20.3%),whereas 4.5% of the cells displayed no staining. In capacitated andionophore-induced acrosome reacted sperm this pattern was reversed,mSLLP1 was localized to the anterior acrosome in only 13.9% ofspermatozoa, whereas mSLLP1 localized to the equatorial region in 70.9%of the cells. The percentage of acrosome reacted (AR) sperm in thepopulation is addressed in the last column.

TABLE 1 Straining Staining in anterior in equatorial No # of AR GroupTreatment/ acrosome segment staining sperm sperm PNA staining (%) (%)(%) counted (%) Non-capacitated/ 70.0 20.3 4.5 177 5.2 acrosome intactPNA(+) Capacitated/ 13.9 70.9 9.3 172 94.1 acrosome reacted PNA(−)Table 2 (comprising Tables 2A and 2B): Effect of SLLP1 anti-serum andrecmSLLP1 on mouse in vitro fertilization using cumulus intact oocytes.In all cases, the sera or the recombinant proteins were present duringfertilization. Two cells embryos were scored as fertilized after 24 h.(*) P 0.05. (A) Decomplemented preimmune (PI) or anti-recmSLLP1 immune(I) sera were added to both gametes 45 min prior to insemination.Statistically significant inhibition was seen at 1:10 and 1:50dilutions. (B) RecmSLLP1 was added to the oocytes 45 min prior toinsemination with untreated capacitated mouse spermatozoa. Significantinhibition was noted at 200 μg/ml of mSLLP1. Control oocytes werepre-incubated with PBS containing no recmSLLP1.

TABLE 2A total # of # of non # of # of 2 cells fertilized % Treatmentexperiments eggs embryo eggs Fertilization PI 1:100 3 20 15 5 75 I 1:1003 20 14 6 70 PI 1:50 3 38 27 11 71 I 1:50 3 46 27 19   59 * PI 1:10 6 6443 21 67 I 1:10 6 80 21 59   26 *

TABLE 2B total # of # of non # of # of 2 cells fertilized % Treatmentexperiments eggs embryo eggs Fertilization Control 3 20 11 9 55recmSLLP1 3 52 25 27 48 50 μg/ml Control 3 29 13 16 45 recmSLLP1 3 64 856  12* 200 μg/ml

Additionally, it can be seen that recombinant mouse SLLP1 canessentially completely inhibit sperm-egg binding, as measured by numberof sperm bound/egg, as well as sperm-egg fusion, relative to thecontrols, including comparison to a recombinant egg protein, ePAD.

Sperm Protein SLLP2: Role in Sperm Binding with Egg and Use to IdentifyEgg Receptors for Sperm.

Human SLLP1 and SLLP2 nucleic acids and proteins were previouslyidentified and sequenced by the inventors (see U.S. patent applicationSer. No. 10/181,611, filed Jul. 18, 2002, the entirety of which isincorporated herein by reference). These proteins were furthercharacterized, and other members of the family were identified by thepresent inventors (see U.S. patent application Ser. No. 10/542,038 filedJul. 13, 2005, the entirety of which is incorporated herein byreference). Both SLLP proteins are sperm specific in their expression.

A schematic comparison of human and mouse SLLP1 and 2 is provided.Additionally, the human SLLP2 cDNA and deduced amino acid sequences weredetermined and are provided. The precursor SLLP2 is about 18 kDA, with apI of 5.9 and the processed form is 15.7 kDA and the pI is 5.9.

Human SLLP1 and SLLP2 each contain a signal peptide. The initial SLLP1polypeptide is synthesized as a 215 amino acid polypeptide having a MWof 23.4 kDa and a pI of 8.0. The mature SLLP1 peptide is 128 amino acidsand has a MW of about 14.6 kDa and pI of 5.0. The initial SLLP2polypeptide is synthesized as a 159 amino acid polypeptide having a MWof 17.9 kDa and a pI of 5.9. The mature SLLP2 peptide is 138 amino acidsand has a MW of about 15.7 kDa and pI of 5.9.

Human SLLP1 and SLLP2 have 48.8% sequence identity between one anotherand have 52% and 44% amino-acid sequence identity with the one knownmature human lysozyme C, respectively, and 44% and 43% amino-acidsequence identity with the predicted lysozyme homologue on chromosome17q11.2. SLLP1 is most closely related to human lysozyme (52% sequenceidentity), whereas SLLP2 is most closely related to chicken lysozyme(51% sequence identity).

The gene encoding SLLP1 is located on Chromosome 17 and is 6012 bp inlength. The SLLP1 gene contains 5 exons (109, 309, 159, 79 and 164 bp,respectively) and 4 introns (3436, 1125, 443 and 188 bp, respectively).The gene encoding SLLP2 is located on Chromosome Xp11.1 and is 1950 bpin length. The SLLP2 gene contains 4 exons (169, 159, 79 and 181 bp,respectively) and 3 introns (428, 830, and 104 bp, respectively).Interestingly, exons 3 and 4 of SLLP1 have a sequence identity withexons 2 and 3 of SLLP2 greater than the overall sequence identitybetween the two complete proteins (i.e. greater than 48.8%) and exons 3and 4 of SLLP1 are identical in size to exons 2 and 3 of SLLP2,respectively.

Mature Mouse SLLP2 cDNA Sequence (SEQ ID NO: 15):AAGATTTATGAACGCTGTGAGCTGGCAAAGAAGCT GGAGGAGGCTGGCCTCGATGGCTTCAAAGGCTATACTGTTGGAGACTGGCTGTGTGTGGCACACTATGAG AGTGGCTTTGACACCTCTTTTGTGGACCACAATCCAGATGGCAGCAGTGAATATGGCATTTTCCAGCTGA ACTCTGCCTGGTGGTGTAACAATGGCATCACACCCACTCAGAACCTCTGCAACATCGATTGTAATGACCT GCTCAACCGCCATATTCTGGATGATATCATATGTGCCAAGAGGGTTGCATCCTCACATAAGAGTATGAAG GCCTGGGATTCCTGGACCCAGCACTGTGCCGGTCATGATTTATCAGAATGGCTAAAGGGGTGTTCTGTGC GTCTGAAAACTGACTCAAGCTATAATAACTGGMature Mouse SLLP2 amino acid Sequence (SEQ ID NO: 16):KIYERCELAKKLEEAGLDGFKGYTVGDWLCVAHYE SGFDTSFVDHNPDGSSEYGIFQLNSAWWCNNGITPTQNLCNIDCNDLLNRHILDDIICAKRVASSHKSMK AWDSWTQHCAGHDLSEWLKGCSVRLKTDSSYNNWHuman SLLP2 cDNA sequence (SEQ ID NO: 17):CTGGGAGGGCTTACAGGTGCCATAATGAAGGCCTG GGGCACTGTGGTAGTGACCTTGGCCACGCTGATGGTTGTCACTGTGGATGCCAAGATCTATGAACGCTGC GAGCTGGCGGCAAGACTGGAGAGAGCAGGGCTGAACGGCTACAAGGGCTACGGCGTTGGAGACTGGCTGT GCATGGCTCATTATGAGAGTGGCTTTGACACCGCCTTCGTGGACCACAATCCTGATGGCAGCAGTGAATA TGGCATTTTCCAACTGAATTCTGCCTGGTGGTGTGACAATGGCATTACACCCACCAAGAACCTCTGCCAC ATGGATTGTCATGACCTGCTCAATCGCCATATTCTGGATGACATCAGGTGTGCCAAGCAGATTGTGTCCT CACAGAATGGGCTTTCTGCCTGGACTTCTTGGAGGCTACACTGTTCTGGCCATGATTTATCTGAATGGCT CAAGGGGTGTGATATGCATGTGAAAATTGATCCAAAAATTCATCCATGACTCAGATTCGAAGAGACAGAT TTTATCTTCCTTTCATTTCTTTCTCTTGTGCATTTAATAAAGGATGGTATCTATAAACAATGC Human SLLP2 Amino acid sequence(SEQ ID NO: 18): MKAWGTVVVTLATLMVVTVDAKIYERCELAARLERAGLNGYKGYGVGDWLCMAHYESGFDTAFVDHNPDG SSEYGIFQLNSAWWCDNGITPTKNLCHMDCHDLLNRHILDDIRCAKQIVSSQNGLSAWTSWRLHCSGHDL SEWLKGCDMHVKIDPKIHP

Proof of the sperm specificity of expression of SLLP2 is provided, asindicated by northern blot analyses of spleen, thymus, prostate, testis,ovary, small intestine, colon, and leukocytes. See also the grid, wherethe square labeled F8 represents testis and the square D12 representsthe gene as cloned and expressed in bacteria.

Recombinant human SLLP2 (“rechSLLP2”) was prepared and expressed in E.coli. Antibodies were prepared against the recombinant SLLP2. It wasthen shown by immuno-electron microscopy that human SLLP2 protein islocalized to the sperm acrosomal region in human sperm. The alignment ofhuman SLLP2 protein with it homologues is shown. Soluble human SLLP2 wasalso isolated and purified, as was mouse SLLP2.

To analyze the biologic activity of human SLLP2, it was determinedwhether it would bind to an egg, in this case, a mouse egg. The resultsof this experiment demonstrate that human SLLP2 can indeed bind withmouse eggs, i.e., cross-species binding.

For comparison to the use of human sperm SLLP2, an experiment wasperformed to determine if purified mouse SLLP2 would bind to Zona intactmouse eggs. The results demonstrate that the sperm protein mSLLP2 doesindeed bind to mouse eggs. The next experiment showed that mouse SLLP2would bind to Zona-free mouse eggs. Competitive assays were thenperformed to determine whether the addition of recombinant mouse SLLP2to a mixture of mouse sperm and mouse eggs could inhibit binding ofsperm and eggs. Various amounts of recombinant mouse SLLP2 were added(5, 25, 50, 100, and 200 μg/ml) and the number of sperm bound per eggwas determined. Relative to BSA (200 μg/ml) or PBS controls, theaddition of recombinant mouse SLLP2 was able to reduce the amount ofbinding occurring between sperm and egg.

Next, it was determined whether recombinant mouse SLLP2 could inhibitmouse sperm-egg fusion. In groups treated with recombinant mouse SLLP2the number of sperm fused per egg was reduced to about 0.1 sperm fusedper egg, at concentrations of 100 or 200 μg/ml recombinant mouse SLLP2,relative to BSA and PBS control treatments.

Next, it was determined whether recombinant mouse SLLP2 could actuallyinhibit or reduce fertilization of mouse eggs with mouse sperm.Recombinant mouse SLLP2 at concentrations of 25, 50, 100, and 200 μg/mlwas incubated with sperm and egg, and compared to BSA and PBS controlsas described above. It can be seen that fertilization occurred at a rateof about 75% in the BSA and PBS controls groups. However, the additionof recombinant mouse (“rm”) SLLP2 inhibited fertilization in adose-dependent fashion, with 200 μg/ml rmSLLP2 nearly totally inhibitingfertilization. Also provided are comparisons of human SLLP2 to othermammalian sequences, and well as a comparison indicating theconservation of SLLP2 ortholog in dogs.

Identification and Characterization of a Novel Mouse Egg Specific TolAProtein (MET)

This novel egg protein was screened through the protein-proteininteraction assay and it was cloned from cDNA library of mouse ovary.This protein appears to be an important pre-patterning protein in mouseeggs and highly regulated in early embryonic development. MET belongs toa TolA (egg specific) family and has a big alanine rich region, which isa feature of this family.

Screening of Putative Egg Receptors for the Mouse Acrosomal SpermProtein SLLP1 Using BIACORE®

BIACORE® systems define the characteristics of proteins in terms oftheir specificity of interaction with other molecules, the rates atwhich they interact (binding and dissociation), and their affinity (howtightly they bind to another molecule).

The application of BIACORE®'s SPR (Surface plasmon resonance) technologyis within the field of proteomics to fish out proteins of interest withsubsequent mass spectrometric identification. Purified recombinant SLLP1was bound on the sensor chip surface and ligand fishing was done withcomplete mouse egg lysate (1000 zona free eggs) bound to SLLP1, withsubsequent identification by mass spectrometry. A number of proteinswere screened with the mass spec data and a novel mouse egg specificTolA-like protein (MET) was selected for further characterization, as itwas a novel TolA protein and EST data base was very specific to egg andpreimplanted embryos. This gene is localized on mouse chromosome number9 and belongs to the TolA family. The TolA family consists of severalbacterial TolA proteins as well as two eukaryotic proteins of currentlyunknown function. In bacteria, Tol proteins are involved in thetranslocation of group A colicins. Colicins are bacterial proteintoxins, which are active against Escherichia coli and other relatedspecies. MET protein is also referred to as a “colicin uptake protein”herein. TolA is anchored to the cytoplasmic membrane by a singlemembrane spanning segment near the N-terminus, leaving most of theprotein exposed to the periplasm.

MET contains a TolA domain and a homologous protein is present inperiplasm of E. coli and that how it's presence in mouse eggs seems tobe important from evolutionary point of view. Bioinformatic analysisshowed that MET has four Protein Kinase C phosphorylation sites, sixCasein Kinase II phosphorylation sites and one Tyrosine Kinasephosphorylation site.

Cloning and Expression of MET

A complete cDNA encoding 440 amino acids was amplified from cDNA library(Ambion) of mouse ovary and cloned in pET expression vector to expressthis protein as a fusion protein in E. coli (BL21) cells. The mRNA wasfound to be 1621 bases long. A splice variant of 416 amino acids wasfound. All the clones were sequenced to check the correct reading frameand a variant was found coding for the same protein with a 24 amino acidfragment deletion. Alignment of the variant with the normal sequence isprovided. The MET nucleic acid (full length/normal and variant)sequences and the protein (full/normal and variant) are as follows:

MET-N Nucleic acid sequence (SEQ ID NO: 1)atggcctctctgaagaggtttcagacgctcgtgcccctggatcacaaacaaggtaccttatttgaaattattggagagcccaagttgcccaagtggttccatgtcgaatgcctggaagatccaaaaagactgtacgtggaacctcggctactggaaatcatgtttggtaaggatggagagcacatcccacatcttgaatctatgttgcacaccctgatacatgtgaacgtgtggggccctgaaaggcgagctgagatttggatattcggaccgccgcctttccgaagggacgttgaccggatgctcactgatctggctcactattgccgcatgaaactgatggaaatagaggctctggaggctggagttgagcgtcgtcgtatggcggcccataaggctgccacccagcctgctcccgtgaaggtccgcgaggctgcccctcggcccgcttccgtgaaggtccctgagacggccacccagcctgctcccgtgaaggtccgcgaggctgcccctcagcccgctccggtgcaggaggtccgcgaggctgcccctcagcaggcttccgtgcaggaggaggtccgcgaggctgccaccgagcaggctcccgtgcaggaggtccgcgaggctgccaccgagcaggctcccgtgcaggaggtcagcgaggctgccaccgagcaggctcccgtgcaggaggtcaacgaggctgccaccgagcaggcttccgtgcaggcggtccgcgaggctgccacccggccggctcccgggaaggtccgcaaggcggccacccagccggctccggtgcaggtttgccaggaggccacccagttggctcccgtgaaggtccgcgaggcggccacccagccggcttccgggaaggtccgcgaggcggccacccagttggctcctgtgaaggtccgcaaggcagccacccagttggctcctgtgaaggtccacgaggcggccacccagccggctccggggaaggtcagcgatgctgccacgcagtcggcttcggtgcaggttcgtgaggctgccacgcagctgtctcccgtggaggccactgatactagccagttggctcaggtgaaggctgatgaagcctttgcccagcacacttcaggggaggcccaccaggttgccaatgggcagtctcccattgaagtctgtgagactgccaccgggcagcattctctagatgtctctagggccttgtcccagaagtgtcctgaggtttttgagtgggagacccagagttgtttggatggcagctatgtcatagttcagcctccaagggat gcctgggaatcatttatcatattaMET-V Nucleic acid sequence (SEQ ID NO: 3)atggcctctctgaagaggtttcagacgctcgtgcccctggatcacaaacaaggtaccttatttgaaattattggagagcccaagttgcccaagtggttccatgtcgaatgcctggaagatccaaaaagactgtacgtggaacctcggctactggaaatcatgtttggtaaggatggagagcacatcccacatcttgaatctatgttgcacaccctgatacatgtgaacgtgtggggccctgaaaggcgagctgagatttggatattcggaccgccgcctttccgaagggacgttgaccggatgctcactgatctggctcactattgccgcatgaaactgatggaaatagaggctctggaggctggagttgagcgtcgtcgtatggcggcccataaggctgccacccagcctgctcccgtgaaggtccgcgaggctgcccctcagcccgctccggtgcaggaggtccgcgaggctgcccctcagcaggcttccgtgcaggaggaggtccgcgaggctgccaccgagcaggctcccgtgcaggaggtccgcgaggctgccaccgagcaggctcccgtgcaggaggtcagcgaggctgccaccgagcaggctcccgtgcaggaggtcaacgaggctgccaccgagcaggcttccgtgcaggcggtccgcgaggctgccacccggccggctcccgggaaggtccgcaaggcggccacccagccggctccggtgcaggtttgccaggaggccacccagttggctcccgtgaaggtccgcgaggcggccacccagccggcttccgggaaggtccgcgaggcggccacccagttggctcctgtgaaggtccgcaaggcagccacccagttggctcctgtgaaggtccacgaggcggccacccagccggctccggggaaggtcagcgatgctgccacgcagtcggcttcggtgcaggttcgtgaggctgccacgcagctgtctcccgtggaggccactgatactagccagttggctcaggtgaaggctgatgaagcctttgcccagcacacttcaggggaggcccaccaggttgccaatgggcagtctcccattgaagtctgtgagactgccaccgggcagcattctctagatgtctctagggccttgtcccagaagtgtcctgaggtttttgagtgggagacccagagttgtttggatggcagctatgtcatagttcagcctccaagggat gcctgggaatcatttatcatattaMET-N Amino Acid Sequence (SEQ ID NO: 2)M A S L K R F Q T L V P L D H K Q G T L F E I I G E P K L P K W F H V EC L E D P K R L Y V E P R L L E I M F G K D G E H I P H L E S M L H T LI H V N V W G P E R R A E I W I F G P P P F R R D V D R M L T D L A H YC R M K L M E I E A L E A G V E R R R M A A H K A A T Q P A P V K V R EA A P R P A S V K V P E T A T Q P A P V K V R E A A P Q P A P V Q E V RE A A P Q Q A S V Q E E V R E A A T E Q A P V Q E V R E A A T E Q A P VQ E V S E A A T E Q A P V Q E V N E A A T E Q A S V Q A V R E A A T R PA P G K V R K A A T Q P A P V Q V C Q E A T Q L A P V K V R E A A T Q PA S G K V R E A A T Q L A P V K V R K A A T Q L A P V K V H E A A T Q PA P G K V S D A A T Q S A S V Q V R E A A T Q L S P V E A T D T S Q L AQ V K A D E A F A Q H T S G E A H Q V A N G Q S P I E V C E T A T G Q HS L D V S R A L S Q K C P E V F E W E T Q S C L D G S Y V I V Q P P R DA W E S F I I L MET-V Amino Acid sequence (SEQ ID NO: 4)M A S L K R F Q T L V P L D H K Q G T L F E I I G E P K L P K W F H V EC L E D P K R L Y V E P R L L E I M F G K D G E H I P H L E S M L H T LI H V N V W G P E R R A E I W I F G P P P F R R D V D R M L T D L A H YC R M K L M E I E A L E A G V E R R R M A A H K A A T Q P A P V K V R EA A P Q P A P V Q E V R E A A P Q Q A S V Q E E V R E A A T E Q A P V QE V R E A A T E Q A P V Q E V S E A A T E Q A P V Q E V N E A A T E Q AS V Q A V R E A A T R P A P G K V R K A A T Q P A P V Q V C Q E A T Q LA P V K V R E A A T Q P A S G K V R E A A T Q L A P V K V R K A A T Q LA P V K V H E A A T Q P A P G K V S D A A T Q S A S V Q V R E A A T Q LS P V E A T D T S Q L A Q V K A D E A F A Q H T S G E A H Q V A N G Q SP I E V C E T A T G Q H S L D V S R A L S Q K C P E V F E W E T Q S C LD G S Y V I V Q P P R D A W E S F I I L

Both MET protein sequences were expressed with the pET vector aftertransformation of bacterial cells with plasmids encoding for fusionproteins, and then induced with 1 mM IPTG(isopropyl-1-thio-β-D-galactopyranoside). The predicted size of the fulllength protein was 48.4 kD, and the predicted size of the variant withthe 24 amino acid deletion was 45.76 kD. Both the expressed and inducedproteins were found running comparatively higher on SDS-PAGE thanpredicted molecular weights, which could be due to post-translationalchanges in the bacterial cells.

MET was found to accumulate in inclusion bodies. Therefore, bacterialcell lysate was used to purify MET with a Nickel-column, as His-tag offusion protein bound to Nickel ions. After purification of MET appearedto be a pure single band on SDS-PAGE. The alanine rich TolA-like domainin MET has been highlighted.

Immuno Characterization of MET in Mouse Eggs

Complete purified MET protein was used to raise antibodies in Guineapigs. Preimmune screening of these Guinea pigs was done with egg lysatefrom 100 mouse eggs. Immunization was done in three animals with aprimary dose of 150 μg of purified protein, and two more booster dosesof 150 μg in three weeks interval. Antibody titer was checked and it wasfound that 50 ng of purified protein was enough to get good signal, evenat 50,000 dilution. At the same time, two animals were used for adjuvantcontrol—which were negative and did not give any signal with purifiedrecombinant MET.

These antibodies were used to characterize the native form of MET inmouse eggs. Western analysis was done with 100 Zona Intact eggs, usingthe above antibodies. One strong signal appeared at its predicted sizeof 48.4 kD, but at the same time one more faint band appeared as it wasin bacterial recombinant of around 65 kD, which proves that the posttranslational changes are different in bacterial recombinant MET and init's native form in the mouse eggs.

Localization of Zinc MET in Mouse Eggs

Expression of MET was found to be localized in mouse ovary sections, andto be very much egg specific protein, without cross-reacting withcumulus cells. Some permeable fixed eggs were also checked for METlocalization and it was observed that MET is abundantly present in theegg cytoplasm. Blastocysts were also checked for MET localization. Itwas found that MET is expressed in the early blastocyst, but thatexpression is reduced in the late blastocyst. Such segregation tospecific blastomeres may be related to pre-patterning.

An experiment was designed to examine MET's role during early embryodevelopment and its localization was studied in all the developmentalstages of in-vitro fertilized eggs through the blastocyst stage. It wasfound that MET is more abundant at the stage of germinal vesicle andgradually reduced after fertilization, but localization seemed to bevery specific at the two cell and four cell stages, and apparentlyappears to be involved in prepatterning and polarity of embryo. However,in the late blastocyst stage it remained only in the peripheral cells,which can be a marker for trophectoderm cells.

Protein-Protein Interaction of MET and SLLP1 (Far-Western Analysis)

MET was discovered from mouse eggs as an interactive partner of Spermacrosomal protein SLLP1, therefore a farwestern analysis was designed toprove the evidence of their binding. To that end, 7 μg of recombinantMET was loaded on 12% SDS-PAGE and transferred to nitrocellulose. Themembrane was overlaid (OL) with recombinant SLLP1 (2 μg/ml) and probedwith anti-SLLP1 monoclonal antibody and secondary antibody. A strongsignal was observed with purified MET, providing further proof that METbinds with SLLP1, and is perhaps an SLLP receptor.

Summary

A full length MET protein and one splice variant were cloned andpurified. This protein is a mammalian egg specific TolA protein, and byEST database was shown only in fertilized and unfertilized eggs.According to the known characteristics of the TolA family, MET has aTolA domain of 241 amino acid residues which is highly Alanine rich.

It is shown herein that MET is abundant in egg cytoplasm, particularlyin cortex. It may segregate to subsets of blastomeres, indicating thatMET may provide evidence of pre-patterning and polarity in the egg.MET's localization with specific expression patterns at differentdevelopmental stages of egg and embryo proved that it is a very stagespecific protein and may be related to pluripotency of the embryoniccells, and later is related to pre-patterning of embryos. Mettranscripts and protein are exclusively oocyte and are preimplantedembryo specific, offers a selective window of targeting as an excellenttarget for contraceptive vaccinogen.

ZEP, a Novel Egg Protein

The following experiments disclose a novel egg surface receptor (calledZEP below) which interacts with an intra-acrosomal protein, SLLP1. Thisnovel egg receptor for SLLP was screened and identified through aprotein-protein interaction assay and was cloned from a mouse ovary cDNAlibrary. This protein appears to be important in sperm binding as wellas in early embryonic development (see below). It belongs to ametalloprotease (egg specific) family and has a specific Zinc bindingsignature. Because it appears to be a Zinc Endopeptidase, it is calledZEP herein.

Further Screening of Putative Egg Receptors which Bind to the IntraAcrosomal Sperm Protein SLLP1 Using BIACORE®

BIACORE® systems can be used to define the characteristics of proteinsin terms of their specificity of interaction with other molecules, therates at which they interact (binding and dissociation), and theiraffinity (how tightly they bind to another molecule).

BIACORE® SPR (Surface plasmon resonance) technology was used to fish outproteins of interest. Purified recombinant SLLP1 was bound on the sensorchip surface and ligand fishing was done with complete mouse egg lysate(1000 zona free eggs) bound to SLLP1, with subsequent identification bymass spectrometry. Proteins were screened with the mass spectrometrydata and ZEP was selected for further characterization as it was a novelmetalloprotease and EST data base was very specific to egg andpreimplanted embryos.

The gene was localized to mouse chromosome number 2 and belongs to theAstacin family. These proteases require zinc for catalysis and membersof this family have an amino terminal propeptide which is cleaved togive the active protease domain.

ZEP showed homology with hatching enzyme EHE7 of Japanese eel Anguillajaponica, therefore it was hypothesized that this protein may beperforming a similar function in mouse embryo development. Bioinformaticanalysis showed that it has 2 glycosylation sites, phosphorylationsites, and myristylation sites; suggestive of a membrane protein. Thisprotein has a typical zinc-binding region signature and that is how itbecomes a zinc-metallopeptidase. Transmembrane topology also predicted astrong transmembrane domain at N-terminal of the protein.

Cloning and Expression of Zinc Endopeptidase (ZEP)

A complete cDNA encoding a 414 amino acid peptide was amplified fromcDNA library (Ambion) of mouse ovary and cloned in pET expression vectorto express this protein as a fusion protein in E. coli (BL21) cells. Allthe clones were sequenced to check the correct reading frame and twomore variants were found coding for the same protein, one with 34 aminoacid deletion and another variant with 34 amino acid deletion and 9amino acid insertion. Alignment of all the variants (normal and twovariants) of Zinc Endopeptidase is given. The mRNA is 2377 nucleotideslong.

All the splice variants were expressed with a pET vector after thetransformation of bacterial cells with the plasmids encoding for fusionproteins, and were then induced with 1 mM IPTG(isopropyl-1-thio-β-D-galactopyranoside). Predicted sizes of normal (N)protein was 45.5 kD, variant-1 (V1) with 34 amino acid deletion and 9amino acid insertion was 43.12 kD and variant-2 (V2) with 34 amino aciddeletion was 41.8 kD. All the expressed and induced proteins were in anSDS-PAGE analysis with their expected size.

This protein was found to accumulate in inclusion bodies. Therefore,bacterial cell lysate was used to purify this protein withNickel-column, as His-tag of fusion protein bound to Nickel ions. Afterpurification of ZEP protein, it was observed that there was an autolyticcleavage and two bands were detected by SDS-PAGE, including a new lowermolecular weight band of about 25 kD. N-terminal sequencing was done onthis lower band and the cleavage site was found after 204 amino acids.It is not yet clear how this protein is cleaved at this particular site,but the sequence data explain the transmembrane domain, zinc-bindingsignature and the cleavage site in the amino acid sequence of ZEP.Additionally, an analysis of the ZEP amino acid sequence furthersuggested a transmembrane structure (as indicated graphically—not shown:TMpred output suggested a transmembrane topology with the preferredmodel comprising the N-terminus outside with one strong transmembranehelices, and a total score: 755 o-i 122-152 (31)).

The nucleic acid and amino acid sequences of the normal (N) ZEP and thetwo variants (V1 and V2) disclosed herein are as follows:

ZEP-N Nucleic acid sequence (SEQ ID NO: 5)atgggagcaccctcagcatccagatgttctggagt ctgcagtaccagtgttccagaaggcttcactcctgagggaagcccggtatttcaggacaaggacatcccc gcaattaaccaagggctcatctcagaggagaccccagaaagcagcttcctggtagaaggggacattatcc ggccaagccctttccgattgttgtcagtgaccaataataaatggcccaagggcgttggtggctttgtgga gatccccttcctgctttccagaaagtatgatgaactcagccgccgggtcattatggatgcctttgctgag tttgaacgtttcacatgcatccggtttgttgcctaccatggtcagagagactttgtttccattcttccta tggcggggtgtttctctggtgtgggacgcagtggagggatgcaggtggtgtccttggcacccacttgtct ccggaagggccgaggcattgtcctacatgagctcatgcacgtacttggcttctggcatgagcattcacgg gcagatcgggaccgctacatccaagtcaactggaacgagatcctcccgggctttgaaatcaacttcatca agtcacggagtaccaatatgttagttccctatgactactcatctgtgatgcattatgggagatttgcctt cagctggcgtgggcagcccaccatcataccactctggacctccagtgttcacattggccagcgatggaac ctgagtacctcagatatcacccgggtctgcaggctgtataactgcagccggagtgtccctgactcccacg ggagagggtttgaggcccagagtgatggaagcagcctcacccctgcctctatatcacgtctacaaagact tctcgaggcactgtcagaggaatctggaagctctgcccctagtggctccaggactggaggccagagtatt gccgggcttggtaacagccagcaaggatgggagcatcctcctcagagcacattcagtgtgggagccttgg caagaccacctcagatgctagccgatgcttcaaaatcggggcctggagcaggtgcagacagcttgtctct agagcagttccagctagcccaggcccccactgtacctcttgctctatttccagaagccagagacaagcca gcacctatccaagatgcctttgagaggctagctccacttccaggaggctgtgcacctggaagtcacatta gagaggtgcccagagacZEP-V1 Nucleic acid sequence (SEQ ID NO: 7)atgggagcaccctcagcatccagatgttctggagt ctgcagtaccagtgttccagaaggcttcactcctgagggaagcccggtatttcaggacaaggacatcccc gcaattaaccaagggctcatctcagaggagaccccagaaagcagcttcctggtagaaggggacattatcc ggccaggggtcagccacggtgtgtctttcccagatgaactcagccgccgggtcattatggatgcctttgc tgagtttgaacgtttcacatgcatccggtttgttgcctaccatggtcagagagactttgtttccattctt cctatggcggggtgtttctctggtgtgggacgcagtggagggatgcaggtggtgtccttggcacccactt gtctccggaagggccgaggcattgtcctacatgagctcatgcacgtacttggcttctggcatgagcattc acgggcagatcgggaccgctacatccaagtcaactggaacgagatcctcccgggctttgaaatcaacttc atcaagtcacggagtaccaatatgttagttccctatgactactcatctgtgatgcattatgggagatttg ccttcagctggcgtgggcagcccaccatcataccactctggacctccagtgttcacattggccagcgatg gaacctgagtacctcagatatcacccgggtctgcaggctgtataactgcagccggagtgtccctgactcc cacgggagagggtttgaggcccagagtgatggaagcagcctcacccctgcctctatatcacgtctacaaa gacttctcgaggcactgtcagaggaatctggaagctctgcccctagtggctccaggactggaggccagag tattgccgggcttggtaacagccagcaaggatgggagcatcctcctcagagcacattcagtgtgggagcc ttggcaagaccacctcagatgctagccgatgcttcaaaatcggggcctggagcaggtgcagacagcttgt ctctagagcagttccagctagcccaggcccccactgtacctcttgctctatttccagaagccagagacaa gccagcacctatccaagatgcctttgagaggctagctccacttccaggaggctgtgcacctggaagtcac attagagaggtgcccagagacZEP-V2 Nucleic acid sequence (SEQ ID NO: 9)atgggagcaccctcagcatccagatgttctggagt ctgcagtaccagtgttccagaaggcttcactcctgagggaagcccggtatttcaggacaaggacatcccc gcaattaaccaagggctcatctcagaggagaccccagaaagcagcttcctgctttccagaaagtatgatg aactcagccgccgggtcattatggatgcctttgctgagtttgaacgtttcacatgcatccggtttgttgc ctaccatggtcagagagactttgtttccattcttcctatggcggggtgtttctctggtgtgggacgcagt ggagggatgcaggtggtgtccttggcacccacttgtctccggaagggccgaggcattgtcctacatgagc tcatgcacgtacttggcttctggcatgagcattcacgggcagatcgggaccgctacatccaagtcaactg gaacgagatcctcccgggctttgaaatcaacttcatcaagtcacggagtaccaatatgttagttccctat gactactcatctgtgatgcattatgggagatttgccttcagctggcgtgggcagcccaccatcataccac tctggacctccagtgttcacattggccagcgatggaacctgagtacctcagatatcacccgggtctgcag gctgtataactgcagccggagtgtccctgactcccacgggagagggtttgaggcccagagtgatggaagc agcctcacccctgcctctatatcacgtctacaaagacttctcgaggcactgtcagaggaatctggaagct ctgcccctagtggctccaggactggaggccagagtattgccgggcttggtaacagccagcaaggatggga gcatcctcctcagagcacattcagtgtgggagccttggcaagaccacctcagatgctagccgatgcttca aaatcggggcctggagcaggtgcagacagcttgtctctagagcagttccagctagcccaggcccccactg tacctcttgctctatttccagaagccagagacaagccagcacctatccaagatgcctttgagaggctagc tccacttccaggaggctgtgcacctggaagtcacattagagaggtgcccagagac ZEP-N Amino acid sequence (SEQ ID NO: 6)M G A P S A S R C S G V C S T S V P E G F T P E G S P V F Q D K D I P AI N Q G L I S E E T P E S S F L V E G D I I R P S P F R L L S V T N N KW P K G V G G F V E I P F L L S R K Y D E L S R R V I M D A F A E F E RF T C I R F V A Y H G Q R D F V S I L P M A G C F S G V G R S G G M Q VV S L A P T C L R K G R G I V L H E L M H V L G F W H E H S R A D R D RY I Q V N W N E I L P G F E I N F I K S R S T N M L V P Y D Y S S V M HY G R F A F S W R G Q P T I I P L W T S S V H I G Q R W N L S T S D I TR V C R L Y N C S R S V P D S H G R G F E A Q S D G S S L T P A S I S RL Q R L L E A L S E E S G S S A P S G S R T G G Q S I A G L G N S Q Q GW E H P P Q S T F S V G A L A R P P Q M L A D A S K S G P G A G A D S LS L E Q F Q L A Q A P T V P L A L F P E A R D K P A P I Q D A F E R L AP L P G G C A P G S H I R E V P R D ZEP-VI Amino acid sequence(SEQ ID NO: 8) M G A P S A S R C S G V C S T S V PE G F T P E G S P V F Q D K D I P A I N Q G L I S E E T P E S S F L V EG D I I R P G V S H G V S F P D E L S R R V I M D A F A E F E R F T C IR F V A Y H G Q R D F V S I L P M A G C F S G V G R S G G M Q V V S L AP T C L R K G R G I V L H E L M H V L G F W H E H S R A D R D R Y I Q VN W N E I L P G F E I N F I K S R S T N M L V P Y D Y S S V M H Y G R FA F S W R G Q P T I I P L W T S S V H I G Q R W N L S T S D I T R V C RL Y N C S R S V P D S H G R G F E A Q S D G S S L T P A S I S R L Q R LL E A L S E E S G S S A P S G S R T G G Q S I A G L G N S Q Q G W E H PP Q S T F S V G A L A R P P Q M L A D A S K P G P G A G A D S L S L E QF Q L A Q A P T V P L A L F P E A R D K P A P I Q D A F E R L A P L P GG C A P G S H I R E V P R D ZEP-V2 Amino acid sequence (SEQ ID NO: 10)M G A P S A S R C S G V C S T S V P E G F T P E G S P V F Q D K D I P AI N Q G L I S E E T P E S S F L L S R K Y D E L S R R V I M D A F A E FE R F T C I R F V A Y H G Q R D F V S I L P M A G C F S G V G R S G G MQ V V S L A P T C L R K G R G I V L H E L M H V L G F W H E H S R A D RD R Y I Q V N W N E I L P G F E I N F I K S R S T N M L V P Y D Y S S VM H Y G R F A F S W R G Q P T I I P L W T S S V H I G Q R W N L S T S DI T R V C R L Y N C S R S V P D S H G R G F E A Q S D G S S L T P A S IS R L Q R L L E A L S E E S G S S A P S G S R T G G Q S I A G L G N S QQ G W E H P P Q S T F S V G A L A R P P Q M L A D A S K S G P G A G A DS L S L E Q F Q L A Q A P T V P L A L F P E A R D K P A P I Q D A F E RL A P L P G G C A P G S H I R E V P R D

Immuno Characterization of Zinc Endopeptidase (ZEP) in Mouse Eggs

Purified protein was used to raise antibodies in Guinea pigs. Preimmunescreening of these Guinea pigs was done with egg lysate of 100 mouseeggs. Immunization was done in three animals with primary dose of 150 μgof purified protein and two more booster doses of 150 μg in three weeksinterval. Antibody titer was checked and it was found that 50 ng ofpurified protein was enough to get good signal even at 50,000 dilution.Concurrently, two animals were used for adjuvant control, which provedto be negative and did not give any signal with purified recombinantZEP.

The antibodies obtained as described were used to characterize thenative form of this protein in mouse eggs. Western analysis was donewith 150 zona intact and 150 zona free eggs, using the above antibodiesas an immune sera screening assay. A signal was found at about 45.5 kD,in both the zona intact and zona free eggs. However, two more bands,which migrated at about 50 kD and 32 kD, were also observed. It ispossible that this protein exists in different forms in the eggs, ordifferent spliced variants may be coding for different sizes of similarproteins.

Localization of Zinc Endopeptidase (ZEP) in Mouse Eggs

It was found that ZEP is egg specific and does not cross react withcumulus cells. Immunolocalization of zinc-peptidase in ovary sections islocalized, and is very much egg specific, including secondary andtertiary follicles. It was further observed that ZEP is localized on theegg surface in the microvillar region. Some blastocysts were alsochecked for the ZEP localization. ZEP is located on the egg surface inthe microvillar region. Also found was a faint signal in the form ofpatches. To determine ZEP's developmental regulation, its localizationwas checked in all the developmental stages of in-vitro fertilized eggsthrough the blastocyst stage and it was found that ZEP is more abundantat the stage of germinal vesicle and gradually reduced afterfertilization. In the blastocyst stage it remained in only some of theperipheral cells.

Protein-Protein Interaction of ZEP and SLLP1

ZEP was picked up from mouse eggs as an interactive partner of the spermacrosomal protein SLLP1. Therefore, to verify the interaction of ZEP andSLLP1 in the native form of the eggs; co-localization was assayed on theegg surface. Mouse eggs were incubated with 10 μg/ml of recombinantSLLP1 for an hour and then with ZEP and SLLP1 antibodies simultaneouslyfor another hour. A secondary antibody of ZEP was cy3 conjugated and asecondary antibody of SLLP1 was FITC conjugated. To comparelocalization, images were captured separately and merged after that. TheZEP signal was present only in the microvillar region, whereas the majorsignal for SLLP1 was located in microvillar region and spread little bitin the perivitelline space. The data demonstrate that the two proteinsare binding with one another, because their signal is completely merged.

To further confirm binding, farwestern analyses were performed tofurther demonstrate that SLLP1 binds with ZEP. To that end, 7.0 μg ofrecombinant ZEP was loaded onto a 12% SDS gel, subjected to PAGE, andtransferred to a nitrocellulose membrane. The membrane was overlaid (OL)with recombinant SLLP1 (2.0 μg/ml) and probed with anti-SLLP1 monoclonalantibody and secondary antibody. A signal was observed with the upperband of ZEP, but not with the lower band. These data suggest that theN-terminal of ZEP has the SLLP1 binding capability, and further suggestwhy it does not bind with the C-terminal band when the N-terminal istruncated.

Summary

The present invention discloses a full length ZEP and two splicevariants. This protein is a mammalian egg specific Zinc Peptidase. Insummary, ZEP is localized at the egg membrane at different developmentalstage. Co-localization with SLLP1 at the egg membrane furtherdemonstrates that it binds with Sperm Acrosomal protein SLLP1. Becauseof the sperm binding capability of ZEP, and its stage specificexpression, this protein seems to be important for fertilization and anexcellent target for contraceptive vaccinogen.

1. A method of inhibiting fertilization comprising: contacting an eggwith a pharmaceutical composition containing an effectivefertilization-inhibiting amount of at least one inhibitor of SAS1R and apharmaceutically-acceptable carrier; wherein said at least one inhibitorof SAS1R is selected from the group consisting of an antibody directedagainst SAS1R and purified SAS1R or a fragment or homolog thereof,wherein the antibody is obtained by immunizing a host animal byinjection with the SAS1R and purified SAS1R or a fragment or homologthereof, wherein SAS1R has a sequence selected from the group consistingof SEQ ID NOs: 6, 8, 10, 19, 20 and 21; wherein the fragment of SAS1Rcomprises amino acids selected from the group consisting of 1-25, 26-50,51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201-225, 226-250,251-275, 276-300, 301-325, 326-350, 351-375, 376-400, and 401-414 ofSAS1R Variant 2 (SEQ ID NO: 6); or selected from the group consisting of1-25, 26-50, 51-75, 76-100, 101-125, 126-150, 151-175, 176-200, 201-225,226-250, 251-275, 276-300, 301-325, 326-350, 351-375, 376-400, 401-425,and 426-435 of SAS1R Variant 1 (SEQ ID NO: 19); and wherein the homologhas at least 90% amino acid sequence identity with SAS1R.
 2. The methodof claim 1, wherein said at least one inhibitor of SAS1R inhibits SAS1Rprotease activity.
 3. The method of claim 1, wherein said at least oneinhibitor of SAS1R inhibits the interaction of SAS1R with a SLLPprotein.
 4. The method of claim 3, wherein said at least one inhibitorof SAS1R is a SAS1R protein, or a fragment or homolog thereof. 5-10.(canceled)
 11. The method of claim 4, wherein at least two differentproteins, or homologs, fragments, or derivatives thereof areadministered.
 12. The method of claim 11, wherein the proteins, orhomologs and fragments, thereof are administered in a mixture ofapproximately equimolar concentrations.
 13. The method of claim 4,wherein the amount of protein, or fragments or homolog thereofadministered is between about 0.001 mg/kg body weight and about 100mg/kg body weight.
 14. The method of claim 1, wherein said method isreversible. 15-16. (canceled)
 17. A method of inducing an immuneresponse in a subject wherein said response is directed to animmunogenic oocyte stage specific metalloprotease sperm proteinreceptor, further wherein inhibition of said metalloprotease inhibitsfertilization, said method comprising administering to the subject apharmaceutical composition comprising an effective immuneresponse-inducing amount of a protein comprising an amino acid sequenceencoding SAS1R or a fragment or homolog thereof, thereby inducing animmune response in a subject.
 18. (canceled)
 19. The method of claim 17,wherein said SAS1R has an amino acid sequence selected from the groupconsisting of SEQ ID NOs:6, 8, 10, 19, 20, 21, and
 23. 20. The method ofclaim 17, said pharmaceutical composition further comprising anadjuvant.
 21. The method of claim 17, said pharmaceutical compositioncomprising more than one SAS1R fragment or homolog thereof.
 22. Themethod of claim 17, wherein said immune response inhibits fertilization.23-33. (canceled)
 34. A contraceptive target comprising an immunogenicoocyte stage specific metalloprotease, wherein: said oocyte specificmetalloprotease functions in fertilization; said oocyte stage specificmetalloprotease is a sperm protein receptor; said oocyte stage specificmetalloprotease is found specifically in secondary and subsequentfollicular stages; inhibition of said target will not affect oocyte stemcells, including naked, primordial, and primary oocytes; and inhibitionof said target inhibits fertilization.
 35. (canceled)
 36. Thecontraceptive target of claim 34, wherein said contraceptive target isSAS1R.
 37. The composition of claim 36, wherein SAS1R has a sequenceselected from the group of amino acid sequences consisting of SEQ ID NOs6, 8, 10, 19, 20, 21, and 23, or a fragment or homolog thereof.
 38. Thecontraceptive target of claim 36, wherein said oocyte stage specificmetalloprotease comprises a sequence selected from the group of aminoacid sequences consisting of SEQ ID Nos: 6, 8, 10, 19, 20, 21, and 23,or a fragment or homolog thereof, interacts with a SLLP protein.
 39. Thecontraceptive target of claim 38, wherein said SLLP protein is SLLP1.40-41. (canceled)
 42. The method of claim 1, wherein the host animal isa rabbit, mouse, or rat.
 43. The method of claim 1, wherein the methodis carried out in vitro.